Aryl acid pyrimidinyl methyl amides, pyridazinyl methyl amides and related compounds

ABSTRACT

The invention provides compounds of Formula (I) that bind to GABA A  receptors. In the above formula, variables are defined herein. Such compounds may be used to modulate ligand binding to GABA A  receptors in vivo or in vitro, and are particularly useful in the treatment of a variety of central nervous system (CNS) disorders in humans, domesticated companion animals, and livestock animals. Compounds provided herein may be administered alone or in combination with one or more other CNS agents to potentiate the effects of the other CNS agent(s). Pharmaceutical compositions and methods for treating such disorders are provided, as are methods for using such ligands for detecting GABA A  receptors (e.g., receptor localization studies)

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application Ser. No.PCT IB2004/000009, filed Feb. 16, 2004, which designates the UnitedStates and which claims priority to U.S. Provisional Application60/448,271, filed Feb. 19, 2003.

FIELD OF THE INVENTION

The present invention relates generally to aryl acid pyrimidinyl methylamides, pyridazinyl methyl amides and related compounds that bind withhigh selectivity and/or high affinity to GABA_(A) receptor. The presentinvention further relates to pharmaceutical compositions comprising suchcompounds and to the use of such compounds in the treatment of centralnervous system (CNS) diseases.

BACKGROUND OF THE INVENTION

The GABA_(A) receptor superfamily represents one of the classes ofreceptors through which the major inhibitory neurotransmitterγ-aminobutyric acid, or GABA, acts. Widely, although unequally,distributed throughout the mammalian brain, GABA mediates many of itsactions through a complex of proteins called the GABA_(A) receptor,which causes alteration in chloride conductance and membranepolarization. A number of drugs, including the anxiolytic and sedatingbenzodiazepines, also bind to this receptor. The GABA_(A) receptorcomprises a chloride channel that generally, but not invariably, opensin response to GABA, allowing chloride to enter the cell. This, in turn,effects a slowing of neuronal activity through hyperpolarization of thecell membrane potential.

GABA_(A) receptors are composed of five protein subunits. A number ofcDNAs for these GABA_(A) receptor subunits have been cloned and theirprimary structures determined. While these subunits share a basic motifof 4 membrane-spanning helices, there is sufficient sequence diversityto classify them into several groups. To date, at least 6α, 3β, 3γ, 1ε,1δ and 2ρ subunits have been identified. Native GABA_(A) receptors aretypically composed of 2 α subunits, 2 β subunits, and 1 γ subunit.Various lines of evidence (such as message distribution, genomelocalization and biochemical study results) suggest that the majornaturally occurring receptor combinations are α₁β₂γ₂, α₂β₃γ₂, α₃β₃γ₂,and α₃β₃γ₂.

The GABA_(A) receptor binding sites for GABA (2 per receptor complex)are formed by amino acids from the α and β subunits. Amino acids fromthe α and γ subunits together form one benzodiazepine site per receptor,at which benzodiazepines exert their pharmacological activity. Inaddition, the GABA_(A) receptor contains sites of interaction forseveral other classes of drugs. These include a steroid binding site, apicrotoxin site, and a barbiturate site. The benzodiazepine site of theGABA_(A) receptor is a distinct site on the receptor complex that doesnot overlap with the site of interaction for other classes of drugs orGABA.

In a classic allosteric mechanism, the binding of a drug to thebenzodiazepine site alters the affinity of the GABA receptor for GABA.Benzodiazepines and related drugs that enhance the ability of GABA toopen GABA_(A) receptor channels are known as agonists or partialagonists, depending on the level of GABA enhancement. Other classes ofdrugs, such as β-carboline derivatives, that occupy the same site andnegatively modulate the action of GABA are called inverse agonists.Those compounds that occupy the same site, and yet have little or noeffect on GABA activity, can block the action of agonists or inverseagonists and are thus referred to as GABA_(A) receptor antagonists.

The important allosteric modulatory effects of drugs acting at thebenzodiazepine site were recognized early, and the distribution ofactivities at different receptor subtypes has been an area of intensepharmacological discovery. Agonists that act at the benzodiazepine siteare known to exhibit anxiolytic, sedative, anticonvulsant and hypnoticeffects, while compounds that act as inverse agonists at this siteelicit anxiogenic, cognition enhancing, and proconvulsant effects.

While benzodiazepines have enjoyed long pharmaceutical use asanxiolytics, these compounds can exhibit a number of unwanted sideeffects such as cognitive impairment, sedation, ataxia, potentiation ofethanol effects, and a tendency for tolerance and drug dependence.Accordingly, there is a need in the art for additional therapeuticagents that modulate GABA_(A) receptor activation and/or activity. Thepresent invention fulfills this need, and provides further relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides compounds that modulate GABA_(A) receptoractivation and/or GABA_(A) receptor-mediated signal transduction. SuchGABA_(A) receptor modulators are preferably high affinity and/or highselectivity GABA_(A) receptor ligands and act as agonists, inverseagonists or antagonists of GABA_(A) receptors, such as human GABA_(A)receptors. As such, they are useful in the treatment of various CNSdisorders.

Within certain aspects, GABA_(A) receptor modulators provided herein arearyl acid pyrimidinyl methyl amides, pyridazinyl methyl amides andrelated compounds of Formula I:

or a pharmaceutically acceptable form thereof, wherein:

-   -   Ar represents phenyl, naphthyl or a 5- to 10-membered heteroaryl        group, each of which is substituted with from 0 to 4 groups        independently selected from R₈;    -   X, Y and Z are:        -   (i) independently nitrogen or CR₁, such that Y is CR₁ if X            is nitrogen; or        -   (ii) Y is taken together with X or Z to form a fused            5-membered heterocyclic ring that is substituted with from 0            to 2 substituents independently chosen from R₁; and the            remainder of X and Z is nitrogen or CR₁;    -   R₁ is independently chosen at each occurrence from:        -   (a) hydrogen, halogen, nitro and cyano; and        -   (b) groups of the formula:

wherein:

-   -   G is a bond, C₁-C₄alkyl, —N(R_(B))—, —O—, —C(═O)—,        —C(═O)N(R_(B))—, —N(R_(B))C(═O)—, —S(O)_(m)—, —CH₂C(═O)—,        —S(O)_(m)N(R_(B))— or —N(R_(B))S(O)_(m)—; wherein m is 0, 1 or        2; and    -   R_(A) and each R_(B) are independently selected from:        -   (i) hydrogen; and        -   (ii) C₁-C₈alkyl, C₂-C₈alkenyl, (C₃-C₈carbocycle)C₀-C₄alkyl            and (3- to 8 membered heterocycle)C₀-C₄alkyl, each of which            is substituted with from 0 to 4 substituents independently            selected from halogen, hydroxy, nitro, cyano, amino,            C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₄alkanoyl, mono- and            di(C₁-C₄alkyl)amino, C₁-C₄haloalkyl and C₁-C₄haloalkoxy;    -   R₄ is hydroxy, nitro, cyano, amino, C₁-C₈alkyl, C₂-C₈alkenyl,        C₂-C₈alkynyl, C₃-C₇cycloalkyl, C₁-C₈haloalkyl, C₁-C₈alkoxy,        C₁-C₈haloalkoxy, C₂-C₈alkyl ether, C₂-C₈ haloalkyl ether, or        mono- or di-(C₁-C₈alkyl)amino(C₀-C₄alkyl);    -   R₅ and R₆ are independently hydrogen, methyl or ethyl;    -   R₇ represents C₁-C₈alkyl, C₂-C₈alkenyl,        (C₃-C₇cycloalkyl)C₀-C₄alkyl, or benzyl that is substituted with        from 0 to 3 substituents independently chosen from halogen,        nitro, trifluoromethyl, trifluoromethoxy, cyano and hydroxy; and    -   R₈ is independently selected at each occurrence from halogen,        hydroxy, nitro, cyano, amino, C₁-C₈ alkyl, C₁-C₈alkenyl,        C₁-C₈alkynyl, (C₃-C₇cycloalkyl)C₀-C₈alkyl, C₁-C₈        haloalkyl,C₁-C₈alkoxy, (C₃-C₇cycloalkyl)C₁-C₈alkoxy,        C₁-C₈haloalkoxy, C₁-C₈alkyl ether, C₁-C₈alkanone, C₁-C₈alkanoyl,        (3- to 7-membered heterocycloalkyl)C₀-C₈alkyl,        C₁-C₈hydroxyalkyl, C₁-C₈aminoalkyl, and mono- and        di-(C₁-C₈alkyl)aminoC₀-C₈alkyl.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising one or more compounds or forms thereof asdescribed above in combination with a pharmaceutically acceptablecarrier, diluent or excipient. Packaged pharmaceutical preparations arealso provided, comprising such a pharmaceutical composition in acontainer and instructions for using the composition to treat a patientsuffering from a CNS disorder such as anxiety, depression, a sleepdisorder, attention deficit disorder or Alzheimer's dementia.

The present invention further provides, within other aspects, methodsfor treating patients suffering from certain CNS disorders, such asanxiety, depression, a sleep disorder, attention deficit disorder,schizophrenia or Alzheimer's dementia, comprising administering to apatient in need of such treatment a GABA_(A) receptor modulatory amountof a compound or form thereof as described above. Methods for improvingshort term memory in a patient are also provided, comprisingadministering to a patient in need of such treatment a GABA_(A) receptormodulatory amount of a compound or form thereof as described above.Treatment of humans, domesticated companion animals (pets) or livestockanimals suffering from certain CNS disorders with an effective amount ofa compound of the invention is encompassed by the present invention.

In a separate aspect, the invention provides methods of potentiating theactions of other CNS active compounds. These methods compriseadministering a GABA_(A) receptor modulatory amount of a compound orsalt of Formula I in conjunction with the administration of another CNSactive compound.

The present invention relates to the use of compounds of Formula I asprobes for the localization of GABA_(A) receptors in sample (e.g., atissue section). In certain embodiments, GABA_(A) receptors are detectedusing autoradiography. Additionally, the present invention providesmethods for determining the presence or absence of GABA_(A) receptor ina sample, comprising the steps of: (a) contacting a sample with acompound as described above under conditions that permit binding of thecompound to GABA_(A) receptor; (b) removing compound that does not bindto the GABA_(A) receptor and (c) detecting a level of compound bound toGABA_(A) receptor.

In yet another aspect, the present invention provides methods forpreparing the compounds disclosed herein, including the intermediates.

These and other aspects of the present invention will become apparentupon reference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of Formula I. Certain preferredcompounds bind to GABA_(A) receptor, preferably with high selectivity;more preferably such compounds further provide beneficial modulation ofbrain function. Without wishing to be bound to any particular theory ofoperation, it is believed that that interaction of such compounds withthe benzodiazepine site of GABA_(A) receptor results in thepharmacological effects of these compounds. Such compounds may be usedin vitro or in vivo to determine the location of GABA_(A) receptors orto modulate GABA_(A) receptor activity in a variety of contexts.

Chemical Description and Terminology

Compounds provided herein are generally described using standardnomenclature. For compounds having asymmetric centers, it should beunderstood that (unless otherwise specified) all of the optical isomersand mixtures thereof are encompassed. All chiral (enantiomeric anddiastereomeric), and racemic forms, as well as all geometric isomericforms of a structure are intended, unless the specific stereochemistryor isomeric form is specifically indicated. Many geometric isomers ofolefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms. Recited compoundsare further intended to encompass compounds in which one or more atomsare replaced with an isotope (i.e., an atom having the same atomicnumber but a different mass number). By way of general example, andwithout limitation, isotopes of hydrogen include tritium and deuteriumand isotopes of carbon include ¹¹C, ¹³C, and ¹⁴C.

Certain compounds are described herein using a general formula thatincludes variables. Unless otherwise specified, each variable withinsuch a formula is defined independently of other variables, and anyvariable that occurs more than one time within a formula is definedindependently at each occurrence. Thus, for example, if a group isdescribed as being substituted with 0-2 R*, then the group may beunsubstituted or substituted with up to two R* groups and R* at eachoccurrence is selected independently from the definition of R*. Inaddition, it will be apparent that combinations of substituents and/orvariables are permissible only if such combinations result in stablecompounds.

The phrase “aryl acid pyrimidinyl methyl amides, pyridazinyl methylamides and related compounds” as used herein, refers to compounds ofFormula I, as well as pharmaceutically acceptable forms thereof.

“Pharmaceutically acceptable forms” of the compounds recited herein arepharmaceutically acceptable salts, hydrates, solvates, crystal forms,polymorphs, chelates, non-covalent complexes, esters, clathrates andprodrugs of such compounds. As used herein, a pharmaceuticallyacceptable salt is an acid or base salt that is generally considered inthe art to be suitable for use in contact with the tissues of humanbeings or animals without excessive toxicity, irritation, allergicresponse, or other problem or complication. Such salts include mineraland organic acid salts of basic residues such as amines, as well asalkali or organic salts of acidic residues such as carboxylic acids.Specific pharmaceutical salts include, but are not limited to, salts ofacids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic,fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic,methanesulfonic, benzene sulfonic, ethane disulfonic,2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric,tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic,succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic,phenylacetic, alkanoic such as acetic, HOOC—(CH₂)_(n)—COOH where n is0-4, and the like. Similarly, pharmaceutically acceptable cationsinclude, but are not limited to sodium, potassium, calcium, aluminum,lithium and ammonium. Those of ordinary skill in the art will recognizefurther pharmaceutically acceptable salts for the compounds providedherein, including those listed by Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). Ingeneral, a pharmaceutically acceptable acid or base salt can besynthesized from a parent compound that contains a basic or acidicmoiety by any conventional chemical method. Briefly, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol or acetonitrileare preferred.

A “prodrug” is a compound that may not fully satisfy the structuralrequirements of Formula I, but is modified in vivo, followingadministration to a patient, to produce a compound of Formula I. Forexample, a prodrug may be an acylated derivative of a compound asprovided herein. Prodrugs include compounds wherein hydroxy, amine orsulfhydryl groups are bonded to any group that, when administered to amammalian subject, cleaves to form a free hydroxyl, amino, or sulfhydrylgroup, respectively. Examples of prodrugs include, but are not limitedto, acetate, formate and benzoate derivatives of alcohol and aminefunctional groups within the compounds provided herein. Prodrugs of thecompounds of Formula I may be prepared, for example, by modifyingfunctional groups present in the compounds in such a way that themodifications are cleaved in vivo to a compound of Formula I.

A “substituent,” as used herein, refers to a molecular moiety that iscovalently bonded to an atom within a molecule of interest. For example,a “ring substituent” may be a moiety such as a halogen, alkyl group,haloalkyl group or other substituent discussed herein that is covalentlybonded to an atom (preferably a carbon or nitrogen atom) that is a ringmember. The term “substituted,” as used herein, means that any one ormore hydrogens on the designated atom is replaced with a selection fromthe indicated substituents, provided that the designated atom's normalvalence is not exceeded, and that the substitution results in a stablecompound (ie., a compound that can be isolated, characterized and testedfor biological activity). When a substituent is oxo (i.e., ═O), then 2hydrogens on the atom are replaced. When aromatic moieties aresubstituted by an oxo group, the aromatic ring is replaced by thecorresponding partially unsaturated ring. For example a pyridyl groupsubstituted by oxo is a pyridone.

The phrase “optionally substituted” indicates that a group may either beunsubstituted or substituted at one or more of any of the availablepositions, typically 1, 2, 3, 4, or 5 positions, by one or more suitablesubstituents such as those disclosed herein. Optional substitution isalso indicated by the phrase “substituted with from 0 to Xsubstituents,” in which X is the maximum number of substituents.

A dash (“—”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —CONH₂ isattached through the carbon atom.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups, and wherespecified, having the indicated number of carbon atoms. Thus, the termC₁-C₆alkyl, as used herein, indicates an alkyl group having from 1 to 6carbon atoms. “C₀-C₄alkyl” refers to a bond or a C₁-C₄alkyl group. Alkylgroups include groups having from 1 to 8 carbon atoms (C₁-C₈alkyl), from1 to 6 carbon atoms (C₁-C₆alkyl) and from 1 to 4 carbon atoms(C₁-C₄alkyl), such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl,2-hexyl, 3-hexyl, and 3-methylpentyl. In certain embodiments, preferredalkyl groups are methyl, ethyl, propyl, butyl, and 3-pentyl.“Aminoalkyl” is an alkyl group as defined herein substituted with one ormore —NH₂ substituents. “Hydroxyalkyl” is an alkyl group as definedherein substituted with one or more —OH substituents.

“Alkenyl” refers to a straight or branched hydrocarbon chain comprisingone or more carbon-carbon double bonds, such as ethenyl and propenyl.Alkenyl groups include C₂-C₈alkenyl, C₂-C₆alkenyl and C₂-C₄alkenylgroups (which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms,respectively), such as ethenyl, allyl or isopropenyl.

“Alkynyl” refers to straight or branched hydrocarbon chains comprisingone or more carbon-carbon triple bonds. Alkynyl groups includeC₂-C₈alkynyl, C₂-C₆alkynyl and C₂-C₄ alkynyl groups, which have from 2to 8, 2 to 6 or 2 to 4 carbon atoms, respectively. Alkynyl groupsinclude for example groups such as ethynyl and propynyl.

By “alkoxy,” as used herein, is meant an alkyl, alkenyl or alkynyl groupas described above attached via an oxygen bridge. Alkoxy groups includeC₁-C₆alkoxy and C₁-C₄alkoxy groups, which have from 1 to 6 or 1 to 4carbon atoms, respectively. Methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy,isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxyare specific alkoxy groups. Similarly “alkylthio” refers to an alkyl,alkenyl or alkynyl group as described above attached via a sulfurbridge.

A “cycloalkyl” is a saturated or partially saturated cyclic group inwhich all ring members are carbon, such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbomyl, adamantyl,decahydro-naphthalenyl, octahydro-indenyl, and partially saturatedvariants of any of the foregoing, such as cyclohexenyl. Such groupstypically contain from 3 to about 10 ring carbon atoms; in certainembodiments, such groups have from 3 to 7 ring carbon atoms (ie.,C₃-C₇cycloalkyl). If substituted, any ring carbon atom may be bonded toany indicated substituent.

In the term “(cycloalkyl)alkyl,” “cycloalkyl” and “alkyl” are as definedabove, and the point of attachment is on the alkyl group. Certain suchgroups are (C₃-C₇cycloalkyl)C₀-C₈alkyl, in which the cycloalkyl group islinked via a direct bond or a C₁-C₈alkyl. This term encompasses, forexample, cyclopropylmethyl, cyclohexylmethyl and cyclohexylethyl.Similarly, “(C₃-C₇cycloalkyl)C₁-C₈alkoxy” refers to a C₃-C₇cycloalkylgroup linked via a C₁-C₈ alkoxy.

The term “alkanoyl” refers to an alkyl group as defined above attachedthrough a carbonyl bridge. Alkanoyl groups include C₂-C₈alkanoyl,C₂-C₆alkanoyl and C₂-C₄alkanoyl groups, which have from 2 to 8, 2 to 6or 2 to 4 carbon atoms, respectively. “C₁alkanoyl” refers to —(C═O)—H,which (along with C₂-C₈alkanoyl) is encompassed by the term“C₁-C₈alkanoyl.” Ethanoyl is C₂alkanoyl.

An “alkanone” is an alkyl group as defined above with the indicatednumber of carbon atoms substituted at least one position with an oxogroup. “C₃-C₈alkanone,” “C₃-C₆alkanone” and “C₃-C₄alkanone” refer to analkanone having from 3 to 8, 6 or 4 carbon atoms, respectively. By wayof example, a C₃ alkanone group has the structure —CH₂—(C═O)—CH₃.

Similarly, “alkyl ether” refers to a linear or branched ethersubstituent linked via a carbon-carbon bond. Alkyl ether groups includeC₂-C₈alkyl ether, C₂-C₆alkyl ether and C₂-C₄alkyl ether groups, whichhave 2 to 8, 6 or 4 carbon atoms, respectively. By way of example, a C₂alkyl ether group has the structure —CH₂—O—CH₃.

“Alkylamino” refers to a secondary or tertiary amine substituent havingthe general structure —NH-alkyl or —N(alkyl)(alkyl), wherein each alkylmay be the same or different. Such groups include, for example, mono-and di-(C₁-C₆alkyl)amino groups, in which each alkyl may be the same ordifferent and may contain from 1 to 6 carbon atoms, as well as mono- anddi-(C₁-C₄alkyl)amino groups. Alkylaminoalkyl refers to an alkylaminogroup linked via an alkyl group (i.e., a group having the generalstructure -alkyl-NH-alkyl or -alkyl-N(alkyl)(alkyl)). Such groupsinclude, for example, mono- and di-(C₁-C₈alkyl)aminoC₁-C₈alkyl, in whicheach alkyl may be the same or different. “Mono- ordi-(C₁-C₈alkyl)aminoC₀-C₄alkyl” refers to a mono- ordi-(C₁-C₈alkyl)amino group linked via a direct bond or a C₁-C₄alkylgroup. The following are representative alkylaminoalkyl groups:

The term “halogen” refers to fluorine, chlorine, bromine and iodine. A“haloalkyl” is a branched or straight-chain alkyl group, substitutedwith 1 or more halogen atoms (e.g., “C₁-C₈haloalkyl” groups have from 1to 8 carbon atoms; “C₁-C₂haloalkyl” groups have from 1 to 2 carbonatoms). Examples of haloalkyl groups include, but are not limited to,mono-, di- or trifluoromethyl; mono-, di- or tri-chloromethyl; mono-,di-, tri-, tetra- or penta-fluoroethyl; and mono-, di-, tri-, tetra- orpenta-chloroethyl. Typical haloalkyl groups are trifluoromethyl anddifluoromethyl. The term “haloalkoxy” refers to a haloalkyl group asdefined above attached via an oxygen bridge. “C₁-C₈haloalkoxy” groupshave from 1 to 8 carbon atoms.

As used herein, the term “aryl” indicates aromatic groups containingonly carbon in the aromatic ring(s). Such aromatic rings may be furthersubstituted with carbon or non-carbon atoms or groups. Typical arylgroups contain 1 to 3 separate, fused, spiro or pendant rings and from 6to about 18 ring atoms, without heteroatoms as ring members.Representative aryl groups include phenyl, naphthyl (including1-naphthyl and 2-naphthyl) and biphenyl, with phenyl preferred incertain embodiments. Bicyclic aryl groups may, but need not, comprise acycloalkyl ring in addition to the aromatic ring (e.g., atetrahydronaphthyl group).

The term “carbocycle” or “carbocyclic group” is used herein to indicatesaturated, partially unsaturated or aromatic groups having 1 ring or 2fused, pendant or spiro rings, with 3 to 8 atoms in each ring, whereinall ring atoms are carbon. A carbocyclic group may be bound through anycarbon atom that results in a stable structure, and may be substitutedon any carbon atom if the resulting compound is stable. Carbocyclicgroups include cycloalkyl and aryl groups. Certain carbocycles recitedherein are (C₃-C₈carbocycle)C₀-C₄alkyl groups (i.e., groups in which a3- to 8-membered carbocyclic group is linked via a direct bond or aC₁-C₄alkyl group).

The term “heterocycle” or “heterocyclic group” is used to indicatesaturated, partially unsaturated, or aromatic groups having 1 or 2rings, with 3 to 8 atoms in each ring, and in at least one ring from 1to 4 independently chosen heteroatoms (i.e., oxygen, sulfur ornitrogen). The heterocyclic ring may be attached via any ring heteroatomor carbon atom that results in a stable structure, and may besubstituted on carbon and/or nitrogen atom(s) if the resulting compoundis stable. Any nitrogen and/or sulfur heteroatoms may optionally beoxidized, and any nitrogen may optionally be quaternized. Certainheterocycles recited herein are (3- to 8-membered heterocycle)C₀-C₄alkylgroups (i.e., groups in which a 3- to 8-membered heterocyclic group islinked via a direct bond or a C₁-C₄alkyl group).

Certain heterocycles are “heteroaryl” (ie., comprise at least onearomatic ring having from 1 to 4 heteroatoms, with the remaining ringatoms being carbon), such as 5- to 7-membered monocyclic groups and 7-to 10-membered bicyclic groups. When the total number of S and O atomsin the heteroaryl group exceeds 1, then these heteroatoms are notadjacent to one another; preferably the total number of S and O atoms inthe heteroaryl group is not more than 1, 2 or 3, more preferably notmore than 1 or 2 and most preferably not more than 1. Examples ofheteroaryl groups include pyridyl, indolyl, pyrimidinyl, pyridazinyl,pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl,isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl and5,6,7,8-tetrahydroisoquinoline. Bicyclic heteroaryl groups may, but neednot, contain a saturated ring in addition to the aromatic ring (e.g., atetrahydroquinolinyl or tetrahydroisoquinolinyl group). A “5- or6-membered heteroaryl” is a monocyclic heteroaryl having 5 or 6 ringmembers. Such 5- or 6-membered heteroaryl groups are preferred incertain embodiments.

Other heterocycles are referred to herein as “heterocycloalkyl” (i.e.,saturated or partially saturated heterocycles). Heterocycloalkyl groupshave from 3 to about 8 ring atoms, and more typically from 3 to 7 or 5to 7 ring atoms. Examples of heterocycloalkyl groups includemorpholinyl, piperazinyl and pyrrolidinyl. A (3- to 7-memberedheterocycloalkyl)C₀-C₈alkyl group is a heterocycloalkyl group havingfrom 3 to 7 ring members that is linked via a direct bond or aC₁-C₈alkyl group. Examples of heterocycloalkyl groups includemorpholinyl, piperazinyl and pyrrolidinyl groups.

The terms “GABA_(A) receptor” and “benzodiazepine receptor” refer to aprotein complex that detectably binds GABA and mediates a dose dependentalteration in chloride conductance and membrane polarization. Receptorscomprising naturally-occurring mammalian (especially human or rat)GABA_(A) receptor subunits are generally preferred, although subunitsmay be modified provided that any modifications do not substantiallyinhibit the receptor's ability to bind GABA (i.e., at least 50% of thebinding affinity of the receptor for GABA is retained). The bindingaffinity of a candidate GABA_(A) receptor for GABA may be evaluatedusing a standard ligand binding assay as provided herein. It will beapparent that there are a variety of GABA_(A)receptor subtypes that fallwithin the scope of the term “GABA_(A) receptor.” These subtypesinclude, but are not limited to, α₂β₃γ₂, α₃β₃γ₂, α₅β₃γ₂ receptors may beobtained from a variety of sources, such as from preparations of ratcortex or from cells expressing cloned human GABA_(A) receptors.Particular subtypes may be readily prepared using standard techniques(e.g., by introducing mRNA encoding the desired subunits into a hostcell, as described herein).

An “agonist” of a GABA_(A) receptor is a compound that enhances theactivity of GABA at the GABA_(A) receptor. Agonists may, but need not,also enhance the binding of GABA to GABA_(A) receptor. The ability of acompound to act as a GABA_(A) agonist may be determined using anelectrophysiological assay, such as the assay provided in Example 4.

An “inverse agonist” of a GABA_(A) receptor is a compound that reducesthe activity of GABA at the GABA_(A) receptor. Inverse agonists, butneed not, may also inhibit binding of GABA to the GABA_(A) receptor. Thereduction of GABA-induced GABA_(A) receptor activity may be determinedfrom an electrophysiological assay such as the assay of Example 4.

An “antagonist” of a GABA_(A) receptor, as used herein, is a compoundthat occupies the benzodiazepine site of the GABA_(A) receptor, but hasno detectable effect on GABA activity at the GABA_(A) receptor. Suchcompounds can inhibit the action of agonists or inverse agonists.GABA_(A) receptor antagonist activity may be determined using acombination of a suitable GABA_(A) receptor binding assay, such as theassay provided in Example 3, and a suitable functional assay, such asthe electrophysiological assay provided in Example 4, herein.

A “GABA_(A) receptor modulator” is any compound that acts as a GABA_(A)receptor agonist, inverse agonist or antagonist. In certain embodiments,such a modulator may exhibit an affinity constant (K_(i)) of less than 1micromolar in a standard GABA_(A) receptor radioligand binding assay, oran EC₅₀ of less than 1 micromolar in an electrophysiological assay asprovided in Example 4. In other embodiments a GABA_(A) receptormodulator may exhibit an affinity constant or EC₅₀ of less than 500 nM,200 nM, 100 nM, 50 nM, 25 nM, 10 nM or 5 nM.

A “GABA_(A) receptor modulatory amount” is an amount of GABA_(A)receptor modulator that, upon administration, results in an effectiveconcentration of modulator at a target GABA_(A)receptor. An effectiveconcentration is a concentration that is sufficient to result in astatistically significant (ie., p≦0.05, which is determined using aconventional parametric statistical analysis method such as a student'sT-test) inhibition of total specific binding of ³H-Flumazenil within theassay described in Example 3.

A GABA_(A) receptor modulator is said to have “high affinity” if theK_(i) at a GABA_(A)receptor is less than 1 micromolar, preferably lessthan 100 nanomolar or less than 10 nanomolar. A representative assay fordetermining K_(i) at GABA_(A) receptor is provided in Example 3, herein.It will be apparent that the K_(i) may depend upon the receptor subtypeused in the assay. In other words, a high affinity compound may be“subtype-specific” (i.e., the K_(i) is at least 10-fold greater for onesubtype than for another subtype). Such compounds are said to have highaffinity for GABA_(A) receptor if the K_(i) for at least one GABA_(A)receptor subtype meets the above criteria.

A GABA_(A) receptor modulator is said to have “high selectivity” if itbinds to a GABA_(A)receptor with a K_(i) that is at least 10-fold lower,preferably at least 100-fold lower, than the K_(i) for binding to othermembrane-bound receptors. In particular, the compound should have aK_(i) that is at least 10-fold greater at the following receptors thanat a GABA_(A) receptor: serotonin, dopamine, galanin, VR1, C₅a, MCH,NPY, CRF, bradykinin and tackykinin. Assays to determine K_(i) at otherreceptors may be performed using standard binding assay protocols, suchas using a commercially available membrane receptor binding assay (e.g.,the binding assays available from MDS PHARMA SERVICES, Toronto, Canadaand CEREP, Redmond, Wash.).

A “CNS disorder” is a disease or condition of the central nervous systemthat is responsive to GABA_(A) receptor modulation in the patient. Suchdisorders include anxiety disorders (e.g., panic disorder, obsessivecompulsive disorder, agoraphobia, social phobia, specific phobia,dysthymia, adjustment disorders, separation anxiety, cyclothymia, andgeneralized anxiety disorder), stress disorders (e.g., post-traumaticstress disorder, anticipatory anxiety acute stress disorder and acutestress disorder), depressive disorders (e.g., depression, atypicaldepression, bipolar disorder and depressed phase of bipolar disorder),sleep disorders (e.g., primary insomnia, circadian rhythm sleepdisorder, dyssomnia NOS, parasomnias including nightmare disorder, sleepterror disorder, sleep disorders secondary to depression, anxiety and/orother mental disorders and substance-induced sleep disorder), cognitivedisorders (e.g., cognition impairment, mild cognitive impairment (MCI),age-related cognitive decline (ARCD), schizophrenia, traumatic braininjury, Down's Syndrome, neurodegenerative diseases such as Alzheimer'sdisease and Parkinson's disease, and stroke), AIDS-associated dementia,dementia associated with depression, anxiety or psychosis, attentiondeficit disorders (e.g., attention deficit disorder and attentiondeficit and hyperactivity disorder), convulsive disorders (e.g.,epilepsy), benzodiazepine overdose and drug and alcohol addiction.

A “CNS agent” is any drug used to treat or prevent a CNS disorder. CNSagents include, for example: serotonin receptor (e.g., 5-HT_(1A))agonists and antagonists and selective serotonin reuptake inhibitors(SSRIs); neurokinin receptor antagonists; corticotropin releasing factorreceptor (CRF₁) antagonists; melatonin receptor agonists; nicotinicagonists; muscarinic agents; acetylcholinesterase inhibitors anddopamine receptor agonists.

A “patient” is any individual treated with a compound provided herein.Patients include humans, as well as other animals such as companionanimals and livestock. Patients may be afflicted with a CNS disorder, ormay be free of such a condition (i.e., treatment may be prophylactic).

Aryl Acid Pyrimidinyl Methyl Amides, Pyridazinly Methyl Amides andRelated Compounds

As noted above, the present invention provides GABA_(A) receptormodulators that are compounds of Formula I, as described above, as wellas pharmaceutically acceptable forms of such compounds. Within certaincompounds of Formula I, Ar is phenyl or pyridyl, each of which issubstituted with from 0 to 4 substituents independently selected fromR₈. In certain embodiments, each R₈ is chosen from halogen, hydroxy,amino, cyano, C₁-C₄alkyl, C₁-C₄alkoxy, mono- and di-C₁-C₄alkylamino,C₂-C₄alkanoyl, (C₃-C₇cycloalkyl)C₀-C₂alkyl, C₁-C₂haloalkoxy andC₁-C₂haloalkoxy. Representative Ar moieties include, for example, phenyland 2-pyridyl, each of which is substituted with from 0 to 3substituents independently chosen from chloro, fluoro, hydroxy, cyano,amino, C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₂alkylamino, C₁-C₂haloalkyl andC₁-C₂haloalkoxy. In certain such compounds, Ar is phenyl or 2-pyridyl,each of which is substituted with 1, 2 or 3 substituents independentlychosen from fluoro and chloro. Such groups include, for example,2,6-difluorophenyl and 6-fluoro-pyrid-2-yl.

R₄, within certain compounds of Formula I, is hydroxy, cyano, amino,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, C₁-C₈alkoxy, C₂-C₈alkyl ether,or mono- or di-(C₁-C₈alkyl) aminoC₀-C₄alkyl. Representative R₄ groupsinclude, for example, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl andC₁-C₆alkoxy.

Within certain compounds provided herein, each R₁ is independentlychosen from:

-   (a) hydrogen and halogen; and-   (b) groups of the formula:

wherein:

G is a bond, —NH—, —N(R_(B))—, —O— or —C(═O)—; and

R_(A) and R_(B) are independently selected from:

-   -   (i) hydrogen; and    -   (ii) C₁-C₆alkyl, C₂-C₆alkenyl and (C₃-C₇cycloalkyl)C₀-C₂alkyl,        each of which substituted with from 0 to 4 substituents        independently selected from hydroxy, halogen, cyano, amino,        C₁-C₂alkyl and C₁-C₂alkoxy.

Representative R₁ groups include, for example, hydrogen, hydroxy,halogen, C₁-C₆alkyl, C₁-C₆alkoxy, C₂-C₄alkanoyl, C₁-C₂haloalkyl,C₁-C₂haloalkoxy, and mono- and di-(C₁-C₄alkyl)amino.

R₅ and R₆, within certain compounds provided herein, are both hydrogen.

R₇, within certain compounds provided herein, is C₃-C₆alkyl, such as3-methyl-butyl, isobutyl or n-butyl.

In certain compounds of Formula I, the variables X, Y and Z areindependently nitrogen or CR₁. In such compounds, the group designated:

may be, for example,

resulting in compounds of Formulas II, III and IV, respectively, inwhich the variables carry the definitions set forth above.

In compounds of Formula II, X, Y and Z are independently CR₁, whereineach R₁ is independently chosen from hydrogen, halogen, C₁-C₆alkyl andC₁-C₆alkoxy; and R₄ is C₁-C₆alkyl or C₁-C₆alkoxy.

In certain compounds of Formula III, the group designated:

wherein R_(1a) and R_(1b) are independently hydrogen, halogen,C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆alkanoyl or C₁-C₆alkoxy.For example, such compounds include those in which R_(1a) is halogen,C₁-C₄alkyl, C₁-C₄alkanoyl or C₁-C₄alkoxy; R_(1b) is hydrogen, methyl ormethoxy; and R₄ is C₁-C₆alkyl or C₁-C₆alkoxy.

In certain compounds of Formula IV, the group designated:

wherein R_(1a) and R_(1b) are independently hydrogen, C₁-C₆alkyl orC₁-C₆alkoxy. For example, such compounds include those in which R_(1a)is hydrogen; R_(1b) is hydrogen, C₁-C₄alkyl and C₁-C₄alkoxy; and R₄ isC₁-C₆alkyl or C₁-C₆alkoxy.

In other compounds of Formula I, Y is taken together with X or Z to forma fused heterocyclic ring. In such compounds, the group designated:

may be, for example,

R₁, within certain such compounds, is independently chosen fromhydrogen, halogen, C₁-C₆alkyl and C₁-C₆alkoxy; and R₄, within certainsuch compounds, is C₁-C₆alkyl or C₁-C₆alkoxy.

Compounds provided herein detectably alter (modulate) ligand binding toGABA_(A) 20 receptor, as determined using a standard in vitro receptorbinding assay. References herein to a “GABA_(A) receptor ligand bindingassay” are intended to refer to the standard in vitro receptor bindingassay provided in Example 3. Briefly, a competition assay may beperformed in which a GABA_(A) receptor preparation is incubated withlabeled (e.g., ³H) ligand, such as Flumazenil, and unlabeled testcompound. Incubation with a compound that detectably modulates ligandbinding to GABA_(A) receptor will result in a decrease or increase inthe amount of label bound to the GABA_(A) receptor preparation, relativeto the amount of label bound in the absence of the compound. Preferably,such a compound will exhibit a K_(i) at GABA_(A) receptor of less than1micromolar, more preferably less than 500 nM, 100 nM, 20 nM or 10 nM.The GABA_(A) receptor used to determine in vitro binding may be obtainedfrom a variety of sources, for example from preparations of rat cortexor from cells expressing cloned human GABA_(A) receptors.

In certain embodiments, preferred compounds have favorablepharmacological properties, including oral bioavailability (such that asub-lethal or preferably a pharmaceutically acceptable oral dose,preferably less than 2 grams, more preferably less than or equal to onegram or 200 mg, can provide a detectable in vivo effect), low toxicity(a preferred compound is nontoxic when a GABA_(A) receptor-modulatoryamount is administered to a subject), minimal side effects (a preferredcompound produces side effects comparable to placebo when a GABA_(A)receptor-modulatory amount of the compound is administered to asubject), low serum protein binding, and a suitable in vitro and in vivohalf-life (a preferred compound exhibits an in vitro half-life that isequal to an in vivo half-life allowing for Q.I.D. dosing, preferablyT.I.D. dosing, more preferably B.I.D. dosing, and most preferablyonce-a-day dosing). Distribution in the body to sites of complementactivity is also desirable (e.g., compounds used to treat CNS disorderswill preferably penetrate the blood brain barrier, while low brainlevels of compounds used to treat periphereal disorders are typicallypreferred).

Routine assays that are well known in the art may be used to assessthese properties, and identify superior compounds for a particular use.For example, assays used to predict bioavailability include transportacross human intestinal cell monolayers, such as Caco-2 cell monolayers.Penetration of the blood brain barrier of a compound in humans may bepredicted from the brain levels of the compound in laboratory animalsgiven the compound (e.g., intravenously). Serum protein binding may bepredicted from albumin binding assays, such as those described byOravcova, et al. (1996) Journal of Chromatography B 677:1-27. Compoundhalf-life is inversely proportional to the frequency of dosage of acompound required to achieve an effective amount. In vitro half-lives ofcompounds may be predicted from assays of microsomal half-life asdescribed by Kuhnz and Gieschen (1998) Drug Metabolism and Disposition26:1120-27.

As noted above, preferred compounds provided herein are nontoxic. Ingeneral, the term “nontoxic” as used herein shall be understood in arelative sense and is intended to refer to any substance that has beenapproved by the United States Food and Drug Administration (“FDA”) foradministration to mammals (preferably humans) or, in keeping withestablished criteria, is susceptible to approval by the FDA foradministration to mammals (preferably humans). In addition, a highlypreferred nontoxic compound generally satisfies one or more of thefollowing criteria: (1) does not substantially inhibit cellular ATPproduction; (2) does not significantly prolong heart QT intervals; (3)does not cause substantial liver enlargement, and (4) does not causesubstantial release of liver enzymes.

As used herein, a compound that “does not substantially inhibit cellularATP production” is a compound that satisfies the criteria set forth inExample 5, herein. In other words, cells treated as described in Example5 with 100 μM of such a compound exhibit ATP levels that are at least50% of the ATP levels detected in untreated cells. In more highlypreferred embodiments, such cells exhibit ATP levels that are at least80% of the ATP levels detected in untreated cells.

A compound that “does not significantly prolong heart QT intervals” is acompound that does not result in a statistically significantprolongation of heart QT intervals (as determined byelectrocardiography) in guinea pigs, minipigs or dogs uponadministration of twice the minimum dose yielding a therapeuticallyeffective in vivo concentration. In certain preferred embodiments, adose of 0.01, 0.05. 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg administeredparenterally or orally does not result in a statistically significantprolongation of heart QT intervals. By “statistically significant” ismeant results varying from control at the p<0.1 level or more preferablyat the p<0.05 level of significance as measured using a standardparametric assay of statistical significance such as a student's T test.

A compound “does not cause substantial liver enlargement” if dailytreatment of laboratory rodents (e.g., mice or rats) for 5-10 days withtwice the minimum dose that yields a therapeutically effective in vivoconcentration results in an increase in liver to body weight ratio thatis no more than 100% over matched controls. In more highly preferredembodiments, such doses do not cause liver enlargement of more than 75%or 50% over matched controls. If non-rodent mammals (e.g., dogs) areused, such doses should not result in an increase of liver to bodyweight ratio of more than 50%, preferably not more than 25%, and morepreferably not more than 10% over matched untreated controls. Preferreddoses within such assays include 0.01, 0.05. 0.1, 0.5, 1, 5, 10, 40 or50 mg/kg administered parenterally or orally.

Similarly, a compound “does not promote substantial release of liverenzymes” if administration of twice the minimum dose yielding atherapeutically effective in vivo concentration does not elevate serumlevels of ALT, LDH or AST in laboratory rodents by more than 100% overmatched mock-treated controls. In more highly preferred embodiments,such doses do not elevate such serum levels by more than 75% or 50% overmatched controls. Alternately, a compound “does not promote substantialrelease of liver enzymes” if, in an in vitro hepatocyte assay,concentrations (in culture media or other such solutions that arecontacted and incubated with hepatocytes in vitro) equivalent totwo-fold the minimum in vivo therapeutic concentration of the compounddo not cause detectable release of any of such liver enzymes intoculture medium above baseline levels seen in media from matchedmock-treated control cells. In more highly preferred embodiments, thereis no detectable release of any of such liver enzymes into culturemedium above baseline levels when such compound concentrations arefive-fold, and preferably ten-fold the minimum in vivo therapeuticconcentration of the compound.

In other embodiments, certain preferred compounds do not inhibit orinduce microsomal cytochrome P450 enzyme activities, such as CYP1A2activity, CYP2A6 activity, CYP2C₉ activity, CYP2C₁₉ activity, CYP2D6activity, CYP2E1 activity or CYP3A4 activity at a concentration equal tothe minimum therapeutically effective in vivo concentration.

Certain preferred compounds are not clastogenic or mutagenic (e.g., asdetermined using standard assays such as the Chinese hamster ovary cellvitro micronucleus assay, the mouse lymphoma assay, the human lymphocytechromosomal aberration assay, the rodent bone marrow micronucleus assay,the Ames test or the like) at a concentration equal to the minimumtherapeutically effective in vivo concentration. In other embodiments,certain preferred compounds do not induce sister chromatid exchange(e.g., in Chinese hamster ovary cells) at such concentrations.

For detection purposes, as discussed in more detail below, compoundsprovided herein may be isotopically-labeled or radiolabeled. Suchcompounds are identical to those described above, but for the fact thatone or more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compoundsprovided herein include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N,¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl. In addition, substitution withheavy isotopes such as deuterium (i.e., ²H) can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas increased in vivo half-life or reduced dosage requirements and,hence, may be preferred in some circumstances.

As noted above, different stereoisomeric forms, such as racemates andoptically active forms, are encompassed by the present invention. Incertain embodiments, it may be desirable to obtain single enantiomers(i.e., optically active forms). Standard methods for preparing singleenantiomers include asymmetric synthesis and resolution of theracemates. Resolution of the racemates can be accomplished byconventional methods such as crystallization in the presence of aresolving agent, or chromatography using, for example, a chiral HPLCcolumn.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising at least one GABA_(A) receptor modulator provided herein,together with at least one physiologically acceptable carrier orexcipient. Such compounds may be used for treating patients in whichGABA_(A) receptor modulation is desirable (e.g., patients undergoingpainful procedures who would benefit from the induction of amnesia, orthose suffering from anxiety, depression, sleep disorders or cognitiveimpairment). Pharmaceutical compositions may comprise, for example,water, buffers (e.g., neutral buffered saline or phosphate bufferedsaline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide,carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, adjuvants, polypeptides or amino acids such as glycine,antioxidants, chelating agents such as EDTA or glutathione and/orpreservatives. Preferred pharmaceutical compositions are formulated fororal delivery to humans or other animals (e.g., companion animals suchas dogs or cats). If desired, other active ingredients may also beincluded, such as additional CNS-active agents.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal, rectalor parenteral administration. The term parenteral as used hereinincludes subcutaneous, intradermal, intravascular (e.g., intravenous),intramuscular, spinal, intracranial, intrathecal and intraperitonealinjection, as well as any similar injection or infusion technique. Incertain embodiments, compositions in a form suitable for oral use arepreferred. Such forms include, for example, tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsion,hard or soft capsules, or syrups or elixirs. Within yet otherembodiments, compositions of the present invention may be formulated asa lyophilizate.

Compositions intended for oral use may further comprise one or morecomponents such as sweetening agents, flavoring agents, coloring agentsand preserving agents in order to provide appealing and palatablepreparations. Tablets contain the active ingredient in admixture withphysiologically acceptable excipients that are suitable for themanufacture of tablets. Such excipients include, for example, inertdiluents (e.g., calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate), granulating and disintegrating agents(e.g., corn starch or alginic acid), binding agents (e.g., starch,gelatin or acacia) and lubricating agents (e.g., magnesium stearate,stearic acid or talc). The tablets may be uncoated or they may be coatedby known techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent(e.g., calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium (e.g., peanut oil, liquid paraffin or olive oil).

Aqueous suspensions comprise the active materials in admixture with oneor more excipients suitable for the manufacture of aqueous suspensions.Such excipients include suspending agents (e.g., sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia);and dispersing or wetting agents (e.g., naturally-occurring phosphatidessuch as lecithin, condensation products of an alkylene oxide with fattyacids such as polyoxyethylene stearate, condensation products ofethylene oxide with long chain aliphatic alcohols such asheptadecaethyleneoxycetanol, condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides such as polyethylene sorbitan monooleate). Aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconutoil) or in a mineral oil such as liquid paraffin. The oily suspensionsmay contain a thickening agent such as beeswax, hard paraffin or cetylalcohol. Sweetening agents such as those set forth above, and/orflavoring agents may be added to provide palatable oral preparations.Such suspension may be preserved by the addition of an anti-oxidant suchas ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, such as sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil (e.g., olive oil orarachis oil) or a mineral oil (e.g., liquid paraffin) or mixturesthereof. Suitable emulsifying agents may be naturally-occurring gums(e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides(e.g., soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol), anhydrides (e.g., sorbitan monoleate) andcondensation products of partial esters derived from fatty acids andhexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate).The emulsions may also contain sweetening and/or flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso comprise one or more demulcents, preservatives, flavoring agentsand/or coloring agents.

A pharmaceutical composition may be prepared as a sterile injectibleaqueous or oleaginous suspension. The compound, depending on the vehicleand concentration used, can either be suspended or dissolved in thevehicle. Such a composition may be formulated according to the known artusing suitable dispersing, wetting agents and/or suspending agents suchas those mentioned above. Among the acceptable vehicles and solventsthat may be employed are water, 1,3-butanediol, Ringer's solution andisotonic sodium chloride solution. In addition, sterile, fixed oils maybe employed as a solvent or suspending medium. For this purpose anybland fixed oil may be employed, including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectible compositions, and adjuvants such as localanesthetics, preservatives and/or buffering agents can be dissolved inthe vehicle.

Pharmaceutical compositions may also be prepared in the form ofsuppositories (e.g., for rectal administration). Such compositions canbe prepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Suitable excipients include, for example, cocoa butter and polyethyleneglycols.

For administration to non-human animals, the composition may also beadded to animal feed or drinking water. It may be convenient toformulate animal feed and drinking water compositions so that the animaltakes in an appropriate quantity of the composition along with its diet.It may also be convenient to present the composition as a premix foraddition to feed or drinking water.

Pharmaceutical compositions may be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of compound following administration). Such formulations maygenerally be prepared using well known technology and administered by,for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Carriers for use within suchformulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofactive compound release. The amount of compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

Compounds provided herein are generally present within a pharmaceuticalcomposition in a therapeutically effective amount. A therapeuticallyeffective amount is an amount that results in a discernible patientbenefit, such as diminution of symptoms of a CNS disorder. A preferredconcentration is one sufficient to inhibit the binding of GABA_(A)receptor ligand to GABA_(A) receptor in vitro. Compositions providingdosage levels ranging from about 0.1 mg to about 140 mg per kilogram ofbody weight per day are preferred (about 0.5 mg to about 7 g per humanpatient per day). The amount of active ingredient that may be combinedwith the carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. Dosage unit forms will generally contain between fromabout 1 mg to about 500 mg of an active ingredient. It will beunderstood, however, that the optimal dose for any particular patientwill depend upon a variety of factors, including the activity of thespecific compound employed; the age, body weight, general health, sexand diet of the patient; the time and route of administration; the rateof excretion; any simultaneous treatment, such as a drug combination;and the type and severity of the particular disease undergoingtreatment. Optimal dosages may be established using routine testing, andprocedures that are well known in the art.

Pharmaceutical compositions may be packaged for treating a CNS disordersuch as anxiety, depression, a sleep disorder, attention deficitdisorder or Alzheimer's dementia. Packaged pharmaceutical preparationsinclude a container holding a therapeutically effective amount of atleast one compound as described herein and instructions (e.g., labeling)indicating that the contained composition is to be used for treating theCNS disorder.

Methods of Use

Within certain aspects, the present invention provides methods forinhibiting the development of a CNS disorder. In other words,therapeutic methods provided herein may be used to treat an existingdisorder, or may be used to prevent, decrease the severity of, or delaythe onset of such a disorder in a patient who is free of detectable CNSdisorder. CNS disorders are discussed in more detail below, and may bediagnosed and monitored using criteria that have been established in theart. Alternatively, or in addition, compounds provided herein may beadministered to a patient to improve short-term memory. Patients includehumans, domesticated companion animals (pets, such as dogs) andlivestock animals, with dosages and treatment regimes as describedabove.

Frequency of dosage may vary, depending on the compound used and theparticular disease to be treated or prevented. In general, for treatmentof most disorders, a dosage regimen of 4 times daily or less ispreferred. For the treatment of sleep disorders a single dose thatrapidly reaches effective concentrations is desirable. Patients maygenerally be monitored for therapeutic effectiveness using assayssuitable for the condition being treated or prevented, which will befamiliar to those of ordinary skill in the art.

Within preferred embodiments, compounds provided herein are used totreat patients in need of such treatment. In general, such patients aretreated with a GABA_(A) receptor modulatory amount of a compound ofFormula I (or a pharmaceutically acceptable form thereof), preferablythe amount is sufficient to alter one or more symptoms of a CNSdisorder. Compounds that act as agonists at α₂β₃γ₂ and α₃β₃γ₂ receptorsubtypes are particularly useful in treat anxiety disorders such aspanic disorder, obsessive compulsive disorder and generalized anxietydisorder; stress disorders including post-traumatic stress, and acutestress disorders. Compounds that act as agonists at α₂β₃γ₂ and α₃β₃γ₂receptor subtypes are also useful in treating anxiety bipolar disorders,schizophrenia and sleep disorders, and may be used in the treatment ofage-related cognitive decline and Alzheimer's disease. Compounds thatact as inverse agonists at the α₅β₃γ₂ receptor subtype or α₁β₂γ₂ andα₅β₃γ₂ receptor subtypes are particularly useful in treating cognitivedisorders including those resulting from Down's Syndrome,neurodegenerative diseases such as Alzheimer's disease and Parkinson'sdisease, and stroke related dementia. Compounds that act as inverseagonists at the α₅β₃γ₂ receptor subtype are particularly useful intreating cognitive disorders through the enhancement of memory, andparticularly short-term memory, in memory-impaired patients; while thosethat act as agonists at the α₅β₃γ₂ receptor subtype are particularlyuseful for the induction of amnesia. Compounds that act as agonists atthe α₁β₂γ₂ receptor subtype are useful in treating convulsive disorderssuch as epilepsy. Compounds that act as antagonists at thebenzodiazepine site are useful in reversing the effect of benzodiazepineoverdose and in treating drug and alcohol addiction.

CNS disorders that can be treated using compounds and compositionsprovided herein include:

-   -   Depression, e.g., depression, atypical depression, bipolar        disorder, depressed phase of bipolar disorder.    -   Anxiety, e.g., general anxiety disorder (GAD), agoraphobia,        panic disorder +/−agoraphobia, social phobia, specific phobia,        Post traumatic stress disorder, obsessive compulsive disorder        (OCD), dysthymia, adjustment disorders with disturbance of mood        and anxiety, separation anxiety disorder, anticipatory anxiety        acute stress disorder, adjustment disorders, cyclothymia.    -   Sleep disorders, e.g., sleep disorders including primary        insomnia, circadian rhythm sleep disorder, dyssomnia NOS,        parasomnias, including nightmare disorder, sleep terror        disorder, sleep disorders secondary to depression and/or anxiety        or other mental disorders, substance induced sleep disorder.    -   Cognition Impairment e.g., cognition impairment, Alzheimer's        disease, Parkinson's disease, mild cognitive impairment (MCI),        age-related cognitive decline (ARCD), stroke, traumatic brain        injury, AIDS associated dementia, and dementia associated with        depression, anxiety and psychosis (including schizophrenia and        hallucinatory disorders).    -   Attention Deficit Disorder. e.g., attention deficit disorder        (ADD), and attention deficit and hyperactivity disorder (ADHD).    -   Speech disorders, e.g., motor tic, clonic stuttering,        dysfluency, speech blockage, dysarthria, Tourette's Syndrome and        logospasm.

Compounds and compositions provided herein can also be used to improveshort-term memory (working memory) in a patient. A therapeuticallyeffective amount of a compound for improving short-term memory loss isan amount sufficient to result in a statistically significantimprovement in any standard test of short-term memory function,including forward digit span and serial rote learning. For example, sucha test may be designed to evaluate the ability of a patient to recallwords or letters. Alternatively, a more complete neurophysicalevaluation may be used to assess short-term memory function. Patientstreated in order to improve short-term memory may, but need not, havebeen diagnosed with memory impairment or considered predisposed todevelopment of such impairment.

In a separate aspect, the present invention provides methods forpotentiating the action (or therapeutic effect) of other CNS agent(s).Such methods comprise administering a GABA_(A)receptor modulatory amountof a compound provided herein in combination with another CNS agent.Such CNS agents include, but are not limited to the following: foranxiety, serotonin receptor (e.g., 5-HT_(1A)) agonists and antagonists;for anxiety and depression, neurokinin receptor antagonists orcorticotropin releasing factor receptor (CRFI) antagonists; for sleepdisorders, melatonin receptor agonists; and for neurodegenerativedisorders, such as Alzheimer's dementia, nicotinic agonists, muscarinicagents, acetylcholinesterase inhibitors and dopamine receptor agonists.Within certain embodiments, the present invention provides a method ofpotentiating the antidepressant activity of selective serotonin reuptakeinhibitors (SSRIs) by administering an effective amount of a GABAagonist compound provided herein in combination with an SSRI. Aneffective amount of compound is an amount sufficient to result in adetectable change in patient symptoms, when compared to a patienttreated with the other CNS agent alone. Combination administration canbe carried out using well known techniques (e.g., as described byDa-Rocha, et al. (1997) J. Psychopharmacology 11(3):211-218; Smith, etal. (1998) Am. J. Psychiatry 155(10):133945; and Le, et al. (1996)Alcohol and Alcoholism 31(suppl.):127-132. See also PCT InternationalPublication Nos. WO 99/47142; WO 99/47171; WO 99/47131 and WO 99/37303.

The present invention also pertains to methods of inhibiting the bindingof benzodiazepine compounds (i.e., compounds that comprise thebenzodiazepine ring structure), such as RO15-1788 or GABA, to GABA_(A)receptor. Such methods involve contacting a GABA_(A)receptor modulatoryamount of a compound provided herein with cells expressingGABA_(A)receptor. This method includes, but is not limited to,inhibiting the binding of benzodiazepine compounds to GABA_(A) receptorsin vivo (e.g., in a patient given an amount of a GABA_(A) receptormodulator provided herein that would be sufficient to inhibit thebinding of benzodiazepine compounds or GABA to GABA_(A) receptor invitro). In one embodiment, such methods are useful in treatingbenzodiazepine drug overdose. The amount of GABA_(A) receptor modulatorthat is sufficient to inhibit the binding of a benzodiazepine compoundto GABA_(A) receptor may be readily determined via a GABA_(A) receptorbinding assay as described in Example 3.

Within separate aspects, the present invention provides a variety of invitro uses for the GABA_(A) receptor modulators provided herein. Forexample, such compounds may be used as probes for the detection andlocalization of GABA_(A) receptors, in samples such as tissue sections,as positive controls in assays for receptor activity, as standards andreagents for determining the ability of a candidate agent to bind toGABA_(A) receptor, or as radiotracers for positron emission tomography(PET) imaging or for single photon emission computerized tomography(SPECT). Such assays can be used to characterize GABA_(A) receptors inliving subjects. Such compounds are also useful as standards andreagents in determining the ability of a potential pharmaceutical tobind to GABA_(A) receptor.

Within methods for determining the presence or absence of GABA_(A)receptor in a sample, a sample may be incubated with a GABA_(A) receptormodulator as provided herein under conditions that permit binding of theGABA_(A) receptor modulator to GABA_(A) receptor. The amount of GABA_(A)receptor modulator bound to GABA_(A) receptor in the sample is thendetected. For example, a GABA_(A) receptor modulator may be labeledusing any of a variety of well known techniques (e.g., radiolabeled witha radionuclide such as tritium, as described herein), and incubated withthe sample (which may be, for example, a preparation of cultured cells,a tissue preparation or a fraction thereof). A suitable incubation timemay generally be determined by assaying the level of binding that occursover a period of time. Following incubation, unbound compound isremoved, and bound compound detected using any method suitable for thelabel employed (e.g., autoradiography or scintillation counting forradiolabeled compounds; spectroscopic methods may be used to detectluminescent groups and fluorescent groups). As a control, a matchedsample may be simultaneously contacted with radiolabeled compound and agreater amount of unlabeled compound. Unbound labeled and unlabeledcompound is then removed in the same fashion, and bound label isdetected. A greater amount of detectable label in the test sample thanin the control indicates the presence of GABA_(A) receptor in thesample. Detection assays, including receptor autoradiography (receptormapping) of GABA_(A) receptors in cultured cells or tissue samples maybe performed as described by Kuhar in sections 8.1.1 to 8.1.9 of CurrentProtocols in Pharmacology (1998) John Wiley & Sons, New York.

For example, GABA_(A) receptor modulators provided herein may be usedfor detecting GABA_(A) receptors in cell or tissue samples. This may bedone by preparing a plurality of matched cell or tissue samples, atleast one of which is prepared as an experimental sample and at leastone of which is prepared as a control sample. The experimental sample isprepared by contacting (under conditions that permit binding ofRO15-1788 to GABA_(A) receptors within cell and tissue samples) at leastone of the matched cell or tissue samples that has not previously beencontacted with any GABA_(A) receptor modulator provided herein with anexperimental solution comprising a detectably-labeled preparation of theselected GABA_(A) receptor modulator at the first measured molarconcentration. The control sample is prepared in the same manner as theexperimental sample and also contains an unlabelled preparation of thesame compound at a greater molar concentration.

The experimental and control samples are then washed to remove unbounddetectably-labeled compound. The amount of remaining bounddetectably-labeled compound is then measured and the amount ofdetectably-labeled compound in the experimental and control samples iscompared. The detection of a greater amount of detectable label in thewashed experimental sample(s) than in control sample(s) demonstrates thepresence of GABA_(A) receptor in the experimental sample.

The detectably-labeled GABA_(A) receptor modulator used in thisprocedure may be labeled with a radioactive label or a directly orindirectly luminescent label. When tissue sections are used in thisprocedure and the label is a radiolabel, the bound, labeled compound maybe detected autoradiographically.

Compounds provided herein may also be used within a variety of wellknown cell culture and cell separation methods. For example, compoundsmay be linked to the interior surface of a tissue culture plate or othercell culture support, for use in immobilizing GABA_(A)receptor-expressing cells for screens, assays and growth in culture.Such linkage may be performed by any suitable technique, such as themethods described above, as well as other standard techniques. Compoundsmay also be used to facilitate cell identification and sorting in vitro,permitting the selection of cells expressing a GABA_(A) receptor.Preferably, the compound(s) for use in such methods are labeled asdescribed herein. Within one preferred embodiment, a compound linked toa fluorescent marker, such as fluorescein, is contacted with the cells,which are then analyzed by fluorescence activated cell sorting (FACS).

Within other aspects, methods are provided for modulating binding ofligand to a GABA_(A) receptor in vitro or in vivo, comprising contactinga GABA_(A) receptor with a sufficient amount of a GABA_(A) receptormodulator provided herein, under conditions suitable for binding ofligand to the receptor. The GABA_(A) receptor may be present insolution, in a cultured or isolated cell preparation or within apatient. Preferably, the GABA_(A) receptor is a present in the brain ofa mammal. In general, the amount of compound contacted with the receptorshould be sufficient to modulate ligand binding to GABA_(A) receptor invitro within, for example, a binding assay as described in Example 3.

Also provided herein are methods for altering the signal-transducingactivity of cellular GABA_(A) receptor (particularly the chloride ionconductance), by contacting GABA_(A) receptor, either in vitro or invivo, with a sufficient amount of a compound as described above, underconditions suitable for binding of Flumazenil to the receptor. TheGABA_(A) receptor may be present in solution, in a cultured or isolatedcell or cell membrane preparation or within a patient, and the amount ofcompound may be an amount that would be sufficient to alter thesignal-transducing activity of GABA_(A) receptor in vitro. In certainembodiments, the amount of compound contacted with the receptor shouldbe sufficient to modulate Flumazenil binding to GABA_(A) receptor invitro within, for example, a binding assay as described in Example 3. Aneffect on signal-transducing activity may be assessed as an alterationin the electrophysiology of the cells, using standard techniques. Theamount of a compound that would be sufficient to alter thesignal-transducing activity of GABA_(A) receptors may be determined viaa GABA_(A) receptor signal transduction assay, such as the assaydescribed in Example 4. The cells expressing the GABA receptors in vivomay be, but are not limited to, neuronal cells or brain cells. Suchcells may be contacted with compounds of the invention through contactwith a body fluid containing the compound, for example through contactwith cerebrospinal fluid. Alteration of the signal-transducing activityof GABA_(A) receptors in cells in vitro may be determined from adetectable change in the electrophysiology of cells expressing GABA_(A)receptors, when such cells are contacted with a compound of theinvention in the presence of GABA.

Intracellular recording or patch-clamp recording may be used toquantitate changes in electrophysiology of cells. A reproducible changein behavior of an animal given a compound of the invention may also betaken to indicate that a change in the electrophysiology of the animal'scells expressing GABA_(A) receptors has occurred.

Preparation of Compounds

Compounds provided herein may generally be prepared using standardsynthetic methods. Starting materials are generally readily availablefrom commercial sources, such as Sigma-Aldrich Corp. (St. Louis, Mo.),or may be prepared as described herein. Representative proceduressuitable for the preparation of compounds provided herein are outlinedin Schemes 1-8, herein, which are not to be construed as limiting theinvention in scope or spirit to the specific reagents and conditionsshown in them. Those having skill in the art, will recognize that thereagents and conditions may be varied and additional steps employed toproduce compounds encompassed by the present invention. In some cases,protection of reactive functionalities may be necessary to achieve thedesired transformations. In general, such need for protecting groups, aswell as the conditions necessary to attach and remove such groups, willbe apparent to those skilled in the art of organic synthesis. Unlessotherwise stated in the schemes below, the variables are as defined inFormula I.

Abbreviations used in Schemes 1-6 and the accompanying Examples are asfollows:

-   -   BF₃—Et₂O trifluoroborane etherate    -   Bu butyl    -   CDCl₃ deuterated chloroform    -   δ chemical shift    -   DCM dichloromethane    -   DMAP N,N-dimethylaminopyridine    -   DME ethylene glycol dimethyl ether    -   DMF N,N-methylformamide    -   EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   Et₃N triethylamine    -   EtOAc ethyl acetate    -   ETOH ethanol    -   HOAc acetic acid    -   HPLC high pressure liquid chromatography    -   H¹ NMR proton nuclear magnetic resonance    -   LiAlH₄ lithium aluminum hydride    -   LC-MS liquid chromatography/mass spectrometry    -   M-CPBA m-chloroperoxybenzoic acid    -   MS mass spectrometry    -   (M+1) mass+1    -   NaOEt sodium ethoxide    -   Pd/C palladium carbon catalyst    -   Pd(PPh₃)₄ tetrakis(triphenylphosphine) palladium (0)    -   Pd₂(dba)₃ tris(dibenzylidineacetone) dipalladium (0)    -   PrI propyl iodine    -   R.T. room temperature    -   SOCl₂ thionyl chloride    -   (t-Bu)₃P tri-t-butyl phosphate    -   THF tetrahydrofuran    -   TLC thin layer chromatography

Scheme 1 illustrates routes to selected compounds of Formula 9. Reactionof 2.5 equivalents of sodium alkoxide with dichloropyridazine ester 1gives the dialkoxy compound 2 (Step 1). Reaction of 1 equivalent sodiumalkoxide with dichloropyridazine ester 1 provides monoalkoxide 3 (Step2). Treatment of monoalkoxide 3 with an appropriate boronic acid or tinreagent under Suzuki or Stille coupling conditions produces a compoundof formula 4 (Step 3). The R₂ group in the boronic acid or tin reagentmay be chosen from a variety of groups including alkyl, alkenyl, aryl,and heterocyclic groups. The ester group in compound 4 is reduced withan appropriate reducing agent to yield alcohol 5 (Step 4). Depending onthe particular nature of 4, a stronger or weaker reducing agent may beselected to facilitate the reaction in Step 4. Thionyl chloride convertsalcohol 5 to chloride 6 (Step 5), which reacts with amines in CH₃CN togive compound 7 (Step 6). Acylation of 7 with an appropriate acylchloride or acid yields compound 9 (Step 8). Alternatively, compounds offormula 9 can be prepared from compounds of formula 8 via Suzuki orStille coupling and acylation (Steps 7 & 8).

Scheme 2 illustrates a method for preparing compounds of Formula 14.Suzuki or Stille coupling of 1 with an appropriate boronic acid or tinreagent produces intermediate 10 in Step 1. Compounds of formula 10 areconverted to formula 11 via a second Suzuki or Stille coupling with aboronic acid or a tin reagent (Step 2). In some cases, in which adesired R₄ group cannot be introduced directly by the coupling reactionof Step 2, additional functional group transformations are employedafter the Step 2 coupling reaction. In general, such transformationswill be apparent to those skilled in the art of organic synthesis.Reduction of 11, followed by thionyl chloride treatment produces acompound of formula 12. Compounds of formula 12 react with aminesfollowed by acylation (Steps 4 and 5) to afford 14.

Scheme 3 illustrates an alternative method for preparing compounds ofFormulas 24 and 25. Pyridazine precursor ketone acid 17 is prepared from15 via alkylation and decarboxylation (steps 1 and 2). 17 reacts withhydrazine (step 3) followed by aromatization with bromine in acetic acid(step 4) to give hydroxxypyridazine 19. 19 is treated with POCl₃ to givechloropyridazine 20 (step 5). N-oxidation of 20 with mCPBA gives 21(step 6), which is converted to chloromethylpyridazine 22 upon treatmentwith POCl₃ (step 7). Amine displacement followed by acylation (steps 8and 9) provides compounds of formula 24. Compound 25 is prepared fromthe reduction of chloropyridazine 24 (step 10).

Scheme 4 illustrates methods for preparing compounds of Formulas 28, 27and 30. Treatment of chloropyridazine 24 with hydrazine followed bytriazole formation with an carboxylic acid gives[1,2,4]triazolo[4,3-b]pyridazines 27 (steps 1 and 2). Compounds 28 areprepared via a palladium coupling procedure with a vinyl tin reagentfollowed by hydrolysis (step 3) from 24. Treatment of acetylpyridazines28 with formamide and formic acid, followed by cyclization upontreatment with POCl₃ produces imidazo[1,5-b]pyridazines 30 (steps 4 and5).

Schemes 5, 6 and 7 illustrate the synthesis of compounds of Formula 43.Alkylation of methyl acetoacetate 31 with an appropriate alkyl iodidegives 32 (Step 1), which reacts with thiourea in the presence of sodiumethoxide to afford 33 (Step 2). Conversion of 33 to 34 is achieved byrefluxing 33 with chloroacetic acid (Step 3). Pyrimidine-2,4-dione 34 istreated with POCl₃ to give 2,4-dichloropyrimidine 35 (Step 4), which ishydrogenated in ethyl acetate in the presence of Pd/C to give aseparable mixture of 36, 37 and 38 (Step 5). The chlorine atom in 37 and38 can be replaced by a nucleophile under either Suzuki/Stille couplingconditions or nucleophilic substitution conditions with alkoxides/amines(Scheme 6). The methyl group in 36, 39 and 40 can be selectivelybrominated to give 41 (Step 1, Scheme 7), which is reacted with amines(Step 2, Scheme 7) followed by acylation to afford compounds 43 (Step 3,Scheme 7).

Scheme 8 illustrates methods for preparing compounds of Formulas 49 and54. Condensation of ketone ester 44 with amidine is achieved bytreatment with excess sodium methoxide in methanol (step 1). Treatmentof 45 with POCl₃ gives the chloro-pyrimidine 46 (step 2). Intermediates46 can be converted to bromomethyl pyrimidine 47 by bromination with Br₂in HOAc at 85° C. (step 3). Alkylation of 47 with an amine (step 4)followed by acylation (step 5) provides compounds of Formula 49.Treatment of chloropyrimidine 46 with hydrazine (step 6) followed byreaction with a carboxylic acid gives triazoles 51 (step 7). Brominationwith bromine in acidic acid selectively occurred on the methyl group togive bromomethyl intermediates 52 (step 8), which are converted to thecompounds of Formula 54 (steps 9 and 10).

EXAMPLES

Starting materials and various intermediates described in the followingExamples may be obtained from commercial sources, prepared fromcommercially available organic compounds, or prepared using knownsynthetic methods. Representative examples of methods suitable forpreparing intermediates of the invention are also set forth below.

In the following Examples, LC-MS conditions for the characterization ofthe compounds herein are:

-   -   1. Analytical HPLC-MS instrumentation: Analyses are performed        using a Waters 600 series pump (Waters Corporation, Milford,        Mass.), a Waters 996 Diode Array Detector and a Gilson 215        auto-sampler (Gilson Inc, Middleton, Wis.), Micromass® LCT        time-of-flight electrospray ionization mass analyzer. Data are        acquired using MassLynx™ 4.0 software, with OpenLynx Global        Server™, Openynxy™, and AutoLynx™ processing.    -   2. Analytical HPLC conditions: 4.6×50 mm, Chromolith SpeedROD        RP-18e column (Merck KGBA, Darmstadt, Germany); UV 10        spectra/sec, 220-340 nm summed; flow rate 6.0 mL/min; injection        volume 1 μl;    -   Gradient conditions—mobile phase A is 95% water, 5% methanol        with 0.05% TFA; mobile phase B is 95% methanol, 5% water with        0.025% TFA, and the gradient is 0-0.5 minutes 10-100% B, hold at        100% B to 1.2 minutes, return to 10% B at 1.21 minutes        inject-to-inject cycle time is 2.15 minutes.    -   3. Analytical MS conditions: capillary voltage 3.5 kV; cone        voltage 30V; desolvation and source temperature are 350° C. and        120° C., respectively; mass range 181-750 with a scan time of        0.22 seconds and an inter scan delay of 0.05 minutes.

Example 1 Synthesis of Representative Aryl Acid Pyridazinyl MethylAmides and Related Compounds

A.N-(4,6-Diethoxy-pyridazin-3-ylmethyl)-2,5-difluoro-N-(3-methyl-butyl)-benzamide

Step 1. Preparation of 4,6-Diethoxy-pyridazine-3-carboxylic Acid EthylEster

Sodium ethoxide (1.71 g, 25 mmol) is added to a stirred solution of4,6-dichloro-pyridazine-3-carboxylic acid ethyl ester (2.20 g, 10 mmol)in THF (35 mL) cooled to 0° C. The reaction mixture is stirred at roomtemperature overnight and then poured into 1N HCl (25 mL). The resultingsolution is then neutralized by saturated NaHCO₃. EtOAc (20 mL) is addedand the layers are separated. The aqueous layer is extracted twice withEtOAc (20 mL) and the combined extracts are washed with brine (25 mL),dried (Na₂SO₄), and evaporated. The residue is purified by flash columnchromatography (eluted with 2:1 Hexane, EtOAc), to give the titleproduct as a light yellow wax. H¹ NMR δ (CDCl3) 6.32 (s, 1H), 4.57 (q,2H, J=7.2 Hz), 4.41 (q, 4H, J=7.2 Hz), 4.09 (q, 4H, J=7.2 Hz), 1.34-1.44(m, 9H). LC-MS (M+1) 241.1

Step 2. Preparation of (4,6-Diethoxy-pyridazin-3-yl)-methanol

LiAlH4 (1N solution in THF, 1 mL) is added to a stirred solution of4,6-diethoxy-pyridazine-3-carboxylic acid ethyl ester (213 mg, 0.8 mmol)in THF (8 mL), cooled to 0° C. The solution is stirred at 0° C. for 3hours. Excess Na₂SO₄.10H₂O is then added and the mixture is stirred atroom temperature for 45 minutes. The solid is filtered and washed withEtOAc. Evaporation of the filtrate in vacuo provides a light yellow oil.This alcohol used in the next step without further purification. LC-MS(M+1) 199.2.

Step 3. Preparation of 3-Chloromethyl-4,6-diethoxy-pyridazine

Excess SOCl₂ is added to a stirred solution of(4,6-diethoxy-pyridazin-3-yl)-methanol (174 mg, 0.88 mmol) in CH₂Cl₂ (4mL). The reaction mixture is stirred at room temperature for 4 hours.The solvent is then removed in vacuo and toluene (4 mL) is added andevaporated to dryness. Flash column chromatography purification (elutedwith 3:1 Hexane, EtOAc) of the residue provides the title product as aclear oil. H¹ NMR δ (CDCl3) 6.28(s, 1H), 4.78 (s, 2H), 4.54 (q, 2H,J=7.2 Hz), 4.11 (q, 2H, J=7.2 Hz), 1.48 (t, 3H, J=7.2 Hz), 1.41 (t,3H,J=7.2 Hz,) LC-MS (M+1) 217.1.

Step 4. Preparation of(4,6-Diethoxy-pyridazin-3-ylmethyl)-(3-methyl-bytyl)-amine

Excess K₂CO₃ is added to a stirred solution of3-chloromethyl-4,6-diethoxy-pyridazine (220 mg, 1.02 mmol) andisoamylamine (440 mg, 5.10 mmol) in acetonitrile (5 mL). The mixture isstirred at room temperature overnight. The solvent is removed in vacuoand water (10 mL) and DCM (15 mL) are added. The layers are separatedand the aqueous layer is extracted with DCM (15 mL). The combinedextracts are washed with brine (10 mL), dried (Na₂SO₄), and evaporatedto provide the title compound as an oil. H¹ NMR δ (CDCl3) 6.22 (s, 2H),4.56 (q, 2H, J=7.2 Hz), 4.12 (q, 2H, J=7.2 Hz), 3.97 (s, 2H), 2.64 (t,2H, J=7.2 Hz), 1.40-1.51 (m, 9H), 0.88 (d, 6H, J=7.2 Hz).

Step 5. Preparation ofN-(4,6-Diethoxy-pyridazin-3-ylmethyl)-2,5-difluoro-N-(3-methyl-butyl)-benzamide

Difluorobenzoic acid chloride (0.11 g) is added dropwise to a stirredsolution of (4,6-diethoxy-pyridazin-3-ylmethyl)-3-methyl-bytyl)-amine(0.16 g) and triethylamine (0.11 g) in DCM (5 ml). The mixture isstirred one hour at room temperature. DCM (10 ml) is added to dilute themixture. The mixture is washed with water (5 mL), dried (Na₂SO₄), andevaporated. Preparative TLC purification of the residue (2:1 of hexane:ethyl acetate) provides the title product (Compound 1). H¹ NMR δ (CDCl3)7.01-7.20 (m,3H), 6.22 (s, 2H), 4.98 (s, 2H), 4.56 (q, 2H, J=7.2 Hz),4.12 (q, 2H, J=7.2 Hz), 3.20 (t, 2H, J=7.2 Hz), 1.35-1.55 (m, 9H), 0.88(d, 6H, J=7.2 Hz). LC-MS (M+1) 408.3.

B. 6-Fluoro-pyridine-2-carboxylic acid(4,6-diethoxy-pyridazin-3-ylmethyl)-(3-methyl-butyl)-amide

EDCI (80 mg) and DMAP (20 mg) are added to a stirred solution of(4,6-diethoxy-pyridazin-3-ylmethyl)-3-methyl-bytyl)-amine (93.5 mg) andfluoropyridinyl acid (59 mg) in DCM (10 ml). The mixture is stirred atroom temperature overnight. DCM (10 ml) is added to dilute the mixture.The mixture is washed with water (5 mL), dried (Na₂SO₄), and evaporated.Preparative TLC purification of the residue (2:1 of hexane: ethylacetate) provides the title product (Compound 2). H¹ NMR 67 CDCl₃)7.80-7.92 (m, 1H), 7.60-7.68 (m, 1H), 6.95-7.02 (m, 1H), 6.22 (s, 2H),4.98 (s, 2H), 4.56 (q, 2H, J=7.2 Hz), 4.12.

C.N-(6-Chloro-4-propyl-pyrimidin-3-ylmethyl)-2,5-difluoro-N-isobutyl-benzamide

Step 1. Preparation of 2-Acetyl-2-propyl-succinic Acid Diethyl Ester

To a solution of 2-acetyl-succinic acid diethyl ester (30 g, 139 mmol)in DMSO (250 ml) is added NaH (5.8 g, 60% in mineral oil, 145 mmol) in10 portions over the period of 1 hour. The resulting solution is stirredat room temperature for another 1.5 hours. PrI (17.1 ml, 174 mmol) isadded slowly over a period of 45 minutes and the resulting solution isstirred at room temperature overnight. Water (500 ml) is added, thesolution is saturated with NaCl and extracted with EtOAc (3×250 ml). Thecombined extracts are washed with brine (400 ml), dried over Na₂SO₄ andevaporated in vacuo. The resulting yellow oil is used for the next stepwithout further purification.

Step 2. Preparation of 3-Acetyl-hexanoic Acid

To 35 g of 2-acetyl-2-propyl-succinic acid diethyl ester, is addedconcentrated HCl (200 ml). The mixture is refluxed (oil bath 105° C.)overnight and to it is added brine (100 ml). The mixture is extractedwith EtOAc (4×150 ml) and the combined extracts are extracted with 2Naqueous NaOH solution (4×100 ml). The NaOH solution is then cooled to 0°C. and acidified with concentrated HCl. The mixture is extracted withEtOAc (4×200 ml) and the combined extracts are washed with brine (200ml), dried (Na₂SO₄) and evaporated in vacuo, which provides the titleproduct as a yellow oil.

Step 3. Preparation of 6-Methyl-5-propyl-4,5-dihydropyridazin-3-one

To a solution of 3-acetyl-hexanoic acid (18.8 g, 119 mmol) in EtOH (150ml) is added NH₂NH₂—H₂O (6.94 ml, 143 mmol) and the mixture is refluxed(oil bath 85° C.) for 4 hours. solvent is removed in vacuo and to theresidue is added water (100 ml) and EtOAc (100 ml). The layers areseparated and the aqueous layer is extracted with EtOAc (3×100 ml). Thecombined extracts are washed with brine (150 ml), dried (Na₂SO₄) andevaporated. The resulting light yellow oil is used without furtherpurification in the next step.

Step 4. Preparation of 6-Methyl-5-propyl-pyridazin-3-one

To a solution of 6-methyl-5-propyl-4,5-dihydropyridazin-3-one (16.7 g,108 mmol) in HOAc (200 ml) heated to 85° C., is added Br₂ (5.5 ml, 108mmol) dropwise. After the addition, the mixture is stirred at 85° C. for1 hour. The solvent is removed in vacuo and the residue is dissolved inEtOAc (250 ml) and washed with NaHCO₃ (200 ml) followed by Na₂S₂O₃saturated solution (50 ml) and brine (200 ml). The organic phase isdried (Na₂SO₄) and evaporated. The resulting yellow solid is used innext step without further purification.

Step 5. Preparation of 6-Chloro-3-methyl-4-propyl-pyridazine

A mixture of 6-methyl-5-propyl-4,5-dipyridazin-3-one (15.3 g, 100 mmol)and POCl₃ (125 ml) is heated at 85° C. for 4 hours. The solvent isremoved and the residue is dissolved in EtOAc (200 ml). The solution iscooled by ice bath and to it is carefully added a saturated aqueoussolution of NaHCO₃ until the aqueous layer becomes basic. The layers areseparated and the aqueous layer is extracted with EtOAc (150 ml). Thecombined organic extracts are washed with brine (150 ml), dried (Na₂SO₄)and evaporated. Flash column separation of the residue with 4:1 hexane,EtOAc provides the title product as a light yellow oil.

Step 6. Preparation of 6-Chloro-3-methyl-4-propyl-pyridazine 2-oxide

To a solution of 6-chloro-3-methyl-4-propyl-pyridazine (8.03 g, 47.06mmol) in CH₂Cl₂ (200 ml) is added mCPBA (11.6 g, 77%, 51.77 mmol). Themixture is stirred at room temperature overnight. Saturated K₂CO₃aqueous solution (50 ml) is added and the layers are separated. Theorganic layer is then washed with brine (100 ml) and dried (Na₂SO₄) andevaporated, which provides the title product as a light yellow oil.

Step 7. Preparation of 6-Chloro-3-chloroethyl-4-propyl-pyridazine

A mixture of 6-chloro-3-methylpropyl-pyridazine 2-oxide (9.3 g, 50 mmol)and POCl₃ (80 ml) is heated at 85° C. for 4 hours. The solvent isremoved and the residue is dissolved in EtOAc (200 ml). The solution iscooled by ice bath and to it is carefully added saturated aqueoussolution of NaHCO₃ until the aqueous layer becomes basic. The layers areseparated and the aqueous layer is extracted with EtOAc (150 ml). Thecombined organic extracts are washed with brine (200 ml), dried (Na2SO₄)and evaporated. Flash column separation of the residue with 5:1 Hexanes,EtOAc provides the title product as a light yellow oil.

Step 8. Preparation of(6-Chloropropyl-pyridazin-3-ylmethyl)-isobutyl-amine

To a solution of 6-chloro-3-chloroethyl-4-propyl-pyridazine (3.4 g,16.58 mmol) in CH₃CN (30 ml) is added K₂CO₃ (9.15 g, 66.3 mmol),isobutylamine (6.6 ml, 66.3 mmol) and the mixture is stirred at roomtemperature overnight. The solvent is removed in vacuo and to theresidue is added water (60 ml) and EtOAc (60 ml). The layers areseparated and the organic layer is washed with brine (20 ml) and dried(Na₂SO₄). Evaporation of the solvent provides a light yellow oil, whichis used to next step without further purification.

Step 9. Preparation ofN-(6-Chloro-4-propyl-pyrimidin-3-ylmethyl)-2,5-difluoro-N-isobutyl-benzamide

To a solution of (6-chloro-4-propyl-pyridazin-3-ylmethyl)-isobutyl-amine(1.47 g, 6.1 mmol) and Et₃N (1.28 ml, 9.2 mmol)in CH₂Cl₂ (30 ml) cooledto 0° C. is added 2,5-difluorobenzoyl chloride (1.14 ml, 9.2 mmol). Themixture is stirred at room temperature overnight. Water (10 ml) is addedand the layers are separated. The organic layer is washed with brine (10ml), then dried (Na₂SO₄) and evaporated. The residue is purified byflash column (silica gel, 1:1 hexane, EtOAc), which provides the titlecompound as light yellow oil. ¹H NMR (CDCl₃) (mixture of rotamers) 7.34(s, 0.8H), 7.24 (s, 0.2H), 7.04-7.11 (m, 3H), 5.05 (s, 1.6H), 4.72 (s,0.4H), 3.08 (d, 2H), 2.74 (t, 1.6H), 2.35 (t, 0.4H), 2.09-2.17 (m, 1H),1.67-1.76 (m, 1.6H0, 1.47-1.51 (m, 0.4H), 1.04 (t, 2.4H), 0.97 (d,1.2H), 0.93 (t, 0.6H), 0.79 (d, 4.8H).

D. 6-Fluoro-pyridine-2-carboxylic acid(6-chloro-4-propyl-pyridazin-3-ylmethyl)-isobutyl-amide

To a stirred solution of(6-chloro-4-propyl-pyridazin-3-ylmethyl)-isobutyl-amine (940 mg, 3.89mmol) and 6-fluoro-pyridine-2-carboxylic acid (663 mg, 4.7 mmol) inCH₂Cl₂ (10 ml) is added EDCI (783 mg, 4.7 mmol) and DMAP (244 mg, 2mmol). The mixture is stirred at room temperature overnight. Water (10ml) is added and the layers are separated. The organic layer is washedwith brine (10 ml), then dried (Na₂SO₄) and evaporated. Flash columnpurification of the residue (2:1 of hexane: ethyl acetate) provides thetitle product. ¹H NMR (CDCl₃) (mixture of rotamers) 7.89 (q, 0.7H), 7.81(q, 0.3H), 7.69 (dd, 0.3H), 7.53 (dd, 0.7H), 7.34 (s, 0.7H), 7.20 (s,0.3H), 6.99 (dd, 0.7H1), 6.91 (dd, 0.3H), 5.19 (s, 0.6H), 5.07 (s,1.4H), 3.41 (d, 0.6H), 3.31 (d, 1.4H1), 2.75 (t, 1.4H1), 2.48 (t, 0.6H),2.08-2.17 (m, 1H), 1.69-1.75 (m, 1.4H1), 1.54-1.60 (m, 0.6H), 1.02 (t,2.11H), 0.99 (d, 1.8H), 0.97 (t, 0.9H), 0.80 (d, 4.2H).

E. 6-Fluoro-pyridine-2-carboxylic acid(4-propyl-pyridazin-3-ylmethyl)-isobutyl-isobutyl-amide

To a solution of 6-fluoro-pyridine-2-carboxylic acid(6-chloro-4-propyl-pyridazin-3-ylmethyl)-isobutyl-amide (110 mg, 0.30mmol) in EtOH (5 ml) is added 10% Pd/C and the mixture is hydrogenatedat 30 psi overnight. The catalyst is filtered and the filtrate isevaporated in vacuo. Preparative TLC separation of the residue with 5%MeOH in CH₂Cl₂ gives the title product as a colorless oil. ¹H NMR(CDCl₃) (mixture of rotamers) 9.02 (d, 0.7H), 8.94 (d, 0.3H), 7.88 (q,0.7H), 7.79 (q, 0.3H), 7.64 (dd, 0.3H), 7.54 (dd, 0.7H), 7.30 (d, 0.7H),7.15 (d, 0.3H), 6.98 (dd, 0.7H), 6.89 (dd, 0.3H), 5.18 (s, 0.6H), 5.14(s, 1.4H), 3.41 (d, 0.6H), 3.29 (d, 1.4H), 2.74 (t, 1.4H), 2.47 (t,0.6H), 2.08-2.18 (m, 1H), 1.66-1.77 (m, 1.4H), 1.51 -1.57 (m, 0.6H),1.00 (t, 2.1H), 0.99 (d, 1.8H), 0.94 (t, 0.9H), 0.79 (d, 4.2H).

F.2,5-Difluoro-N-isobutyl-N-(7-propyl-[1,2,4]triazolo[4,3-B]pyridazin-6-ylmethyl)-benzamide

Step 1. Preparation of2,5-Difluoro-N-(6-hydrazino-4-propyl-pyridazin-3-ylmethyl)-N-isobutyl-benzamide

To a solution ofN-(6-chloro-4-propyl-pyrimidin-3-ylmethyl)-2,5-difluoro-N-isobutyl-benzamide(188 mg, 0.52 mmol) in EtOH (5 ml) is added hydrazine monohydrate (91mg, 1.82 mmol) and the mixture is heated at 85° C. overnight. Thesolvent is removed in vacuo and to the residue is added EtOAc (10 ml)and water (10 ml). The layers are separated and the aqueous layer isextracted with EtOAc (2×10 ml). The combined extracts are washed withbrine (10 ml), dried (Na₂SO₄) and evaporated in vacuo, which providesthe title compound as a light yellow oil.

Step 2. Preparation of2,5-Difluoro-N-isobutyl-N-(7-propyl-[1,2,4]triazolo[4,3-b]pyridazin-6-ylmethyl)-benzamide

A solution of2,5-difluoro-N-(6-hydrazino-4-propyl-pyridazin-3-ylmethyl)-N-isobutyl-benzamide(78 mg, 0.21 mmol) in HCOOH (3 ml) is heated at 110° C. overnight. Thesolvent is removed in vacuo and to the residue is added EtOAc (10 ml)and saturated NaHCO₃ aqueous solution (10 ml). The layers are separatedand the aqueous layer is extracted with EtOAc (10 ml). The combinedextracts are washed with brine (10 ml), dried (Na₂SO₄) and evaporated invacuo. Preparative TLC separation of the residue with 5% MeOH in CH₂Cl₂provides the title compound as a light yellow oil. ¹H NMR (CDCl3)(mixture of rotamers) 9.07 (s, 0.2H), 9.00 (s, 0.8H), 7.87 (s, 0.8H),7.78 (s, 0.2H), 6.96-7.13 (m, 3H), 4.91 (s, 1.6H), 4.62 (s, 0.4H), 3.13(d, 2H), 2.71 (t, 1.6H), 2.40 (t, 0.4H), 2.09-2.13 (m, 0.2H), 1.92-2.00(m, 0.8H) 1.74-1.83 (m, 1.6H), 1.48-1.55 (m, 0.4H), 1.09 (t, 2.4H), 1.00(d, 1.2 H), 0.96 (t, 0.6H), 0.81 (d, 4.8H).

G.2,5-Difluoro-N-isobutyl-N-(3-methyl-7-propyl-[1,2,4]triazolo[4,3-B]pyridazin-6-ylmethyl)-benzamide

A solution of2,5-difluoro-N-(6-hydrazinopropyl-pyridazin-3-ylmethyl)-N-isobutyl-benzamide(63 mg, 0.17 mmol) in HOAc (3 ml) is heated at 110° C. overnight. Thesolvent is removed in vacuo and to the residue is added EtOAc (10 ml)and saturated NaHCO₃ aqueous solution (10 ml). The layers are separatedand the aqueous layer is extracted with EtOAc (10 ml). The combinedextracts are washed with brine (10 ml), dried (Na₂SO₄) and evaporated invacuo. Preparative TLC separation of the residue with 5% MeOH in CH₂Cl₂provides the title compound as a light yellow oil. ¹H NMR (CDCl₃)(mixture of rotamers) 7.79 (s, 0.75H), 7.71 (s, 0.25H), 6.95-7.12 (m,3H), 4.91 (s, 1.5H), 4.62 (s, 0.5H), 3.17 (d, 2H), 2.81 (s, 0.75H), 2.79(s, 2.25H), 2.69 (t, 1.5H), 2.39 (t, 0.5H), 2.06-2.17 (m, 0.25H),1.91-2.01 (m, 0.75H), 1.72-1.82 (m, 1.5H), 1.45-1.55 (m, 0.5H), 1.08 (t,2.25H), 1.00 (d, 4.5H), 0.93 (t, 0.75H), 0.82 (d, 1.5H).

H.N-(6-Acetyl-4-propyl-pyridazin-3-ylmethyl)-2,5-difluoro-N-isobutyl-benzamide

A solution ofN-(6-chloro-4-propyl-pyrimidin-3-ylmethyl)-2,5-difluoro-N-isobutyl-benzamide(270 mg, 0.71 mmol), tributyl(1-ethyoxyvinyl)tin (386 mg, 1.07 mmol) andPd(PPh₃)₂Cl₂ (49 mg, 0.07 mmol) in toluene (8 ml) is degassed by Ar for10 minutes and then heated at 110° C. in a seared tube overnight. Thesolvent is evaporated in vacuo and to the residue is added THF (3 ml)and 3N HCl (3 ml), and the mixture is stirred at R.T. for 2 hours.Saturated NaHCO₃ aqueous solution is added to neutralize the reactionmixture. EtOAc (10 ml) is added and the layers are separated. Theaqueous layer is extracted with EtOAc (10 ml) and the combined extractsare washed with brine (10 ml), dried (Na₂SO₄) and evaporated. Flashcolumn separation of the residue with 2:1 EtOAc, hexane gives the titlecompound as a light yellow oil. ¹H NMR (CDCl₃) (mixture of rotamers)7.95 (s, 0.8H), 7.84 (s, 0.2H), 7.01-7.13 (m, 3H), 5.11 (s, 1.6H), 4.80(s, 0.4H), 3.17 (d, 2H), 2.89 (s, 3H), 2.81 (t, 2.4H), 2.39 (t, 0.6H),2.07-2.18 (m, 1H), 1.70-1.79 (m, 1.6H), 1.48-1.53 (m, 0.4H), 1.04 (t,2.4H), 0.99 (d, 1.2H), 0.90 (t, 0.6H), 0.81 (d, 4.8H).

I.2,5-Difluoro-N-isobutyl-N-(5-methyl-3-propyl-imidazo[1,5-B]pyridazin-2-ylmethyl)-benzamide

Step 1. Preparation of2,5-Difluoro-N-[6-(1-formylamino-ethyl)-4-propyl-pyridazin-3-ylmethyl]-N-isobutyl-benzamide

To HCONH₂ (0.5 ml) heated to 160° C. is added a solution ofN-(6-acetyl4-propyl-pyridazin-3-ylmethyl)-2,5-difluoro-N-isobutyl-benzamide(103 mg, 0.26 mmol) and HCOOH (0.2 ml) in HCONH₂ (0.5 ml). The mixtureis heated at 160° C. for 30 minutes. Additional 0.2 ml of HCOOH is addedevery 20 minutes for 3 times, at which time LC-MS analysis shows nostarting material. The mixture is cooled to R.T. and saturated NaHCO₃aqueous solution is added to neutralize the reaction mixture. EtOAc (10ml) is added and the layers are separated. The aqueous layer isextracted with EtOAc (10 ml) and the combined extracts are washed withbrine (10 ml), dried (Na₂SO₄) and evaporated. Flash column separation ofthe residue with 3:1 EtOAc, hexane gives the title compound as a lightyellow oil.

Step 2. Preparation of2,5-Difluoro-N-isobutyl-N-(5-methyl-3-propyl-imidazo[1,5-b]pyridazin-2-ylmethyl)-benzamide

A mixture of2,5-difluoro-N-[6-(1-formylamino-ethyl)-4-propyl-pyridazin-3-ylmethyl]-N-isobutyl-benzamide(89 mg) and POCl₃ (3 ml) is heated at 100° C. for 3 hours. The solventis removed in vacuo and to the residue is added saturated NaHCO₃ aqueoussolution to neutralize the reaction mixture. EtOAc (10 ml) is added andthe layers are separated. The aqueous layer is extracted with EtOAc (10ml) and the combined extracts are washed with brine (10 ml), dried(Na₂SO₄) and evaporated. Flash column separation of the residue with 5%MeOH in CH₂Cl₂ gives the title compound as a light yellow oil. ¹H NMR(CDCl₃) (mixture of rotamers) 8.30 (s, 0.4H), 8.25 (s, 0.6H), 7.45 (s,0.6H), 7.35 (s, 0.4H), 7.00-7.13 (m, 3H), 4.83 (s, 1.2H), 4.48 (s,0.8H), 3.08 (d, 2H), 2.48 (s, 1.8H), 2.46 (s, 1.2H), 2.56 (t, 1.2H),2.08-2.17 (m, 0.4H), 1.95-2.05 (m, 0.6H), 2.26 (t, 0.8H), 1.66-1.75 (m,1.2H), 1.38-1.47 (m, 0.8H), 1.05 (t, 1.8H), 0.99 (d, 2.4H), 0.91 (t,1.2H), 0.79 (d, 3.6H).

Example 2 Synthesis of Additional Representative Aryl Acid PyrimidinylMethyl Amides

A. 6-Fluoro-pyridine-2-carboxylic acid(6-chloro-5-propyl-pyrimidin-4-ylmethyl)-(3-methyl-butyl)-amide

Step 1. Preparation of 2-acetyl-pentanoic Acid Methyl Ester

A solution of methyl acetoacetate (10.8 ml, 100 mmol) in DME (50 ml) isadded dropwise to a suspension of NaH (95% dry, 2.44 g, 100 mmol) in DME(250 ml) cooled to 0° C. The resulting solution is stirred at roomtemperature for 1 hour. Bu₄NI (3.7 g, 10 mmol) is added followed by PrI.The mixture is then stirred at reflux for 6 hours. The solvent isremoved in vacuo and water (200 ml) and EtOAc (200 ml) are added. Thelayers are separated and the aqueous layer is extracted with EtOAc (200ml). The combined extracts are washed with brine (200 ml) and dried(Na₂SO₄). Evaporation of the solvent provides a light yellow oil. Flashcolumn chromatography of the residue by silica gel, eluting with 7:1hexane, EtOAc provides the title product as a colorless oil. LC-MS, M+1159.2

Step 2. Preparation of6-methyl-5-propyl-2-thio-2,3-dihydropyrimidin-4-one

A mixture of 2-acetyl-pentanoic acid methyl ester (3.0 g, 19 mmol),thiourea (7.23 g, 95 mmol), and NaOEt (7.76 g, 114 mmol) in EtOH (50 ml)is stirred at reflux for 4 hours. Solvent is removed in vacuo and theresidue is dissolved in water (40 ml). The solution is carefullyacidified to pH 4 with concentrated HCl and stirred at 0° C. for 45minutes. The solid which forms is filtered, washed with water and dried,to provide the title compound as a light yellow solid. LC-MS, M+1 185.1

Step 3. Preparation of 6-methyl-5-propyl-pyrimidin-2,4-dione

A 10% aqueous solution of chloroacetic acid (40 ml) is added to6-methyl-5-propyl-2-thio-2,3-dihydropyrimidin-4-one (1.74 g, 9.4 mmol).The mixture is heated at reflux for 4 hours and then cooled in an icebath. The solid which forms is collected by filtration, washed withwater, and dried, to provide a white solid. LC-MS, M+1 169.2

Step 4. Preparation of 2,4-dichloro-6-methyl-5-propyl-pyrimidine

A mixture of 6-methyl-5-propyl-pyrimidin-2,4-dione (1.68 g, 10 mmol),POCl₃ (10 ml), and DMF (3 drops) is stirred at 85° C. for 4 hours. Thesolvent is removed in vacuo and EtOAc (20 ml) and water (20 ml) areadded to the residue. The layers are separated and the aqueous layer isextracted with EtOAc (20 ml). The combined extracts are washed withbrine (20 ml) and dried (Na₂SO₄). Evaporation of the solvent provides alight yellow oil, which is used for next step without furtherpurification. LC-MS, M+1 206.2

Step 5. Hydrogenation of 2,4-Dichloro-6-methyl-5-propyl-pyrimidine

5% Pd/C (25 mg) and CH₃COONa (820 mg, 10 mmol) are added to a solutionof 2,4-dichloro-6-methyl-5-propyl-pyrimidine (1.02 g, 5 mmol) in EtOAc(25 ml). The mixture is then hydrogenated at 50 psi overnight. Thecatalyst is filtered and the solvent is removed in vacuo. Flash columnchromatography of the residue on silica gel by 4:1 hexane, EtOAcprovides 2-chloro-6-methyl-5-propyl-pyrimidine (LC-MS, M+1 171.7),4-chloro-6-methyl-5-propyl-pyrimidine (LC-MS, M+1 171.7) and6-methyl-5-propyl-pyrimidine (LC-MS, M+1 157.7) as 1:1:1.5 mixture,which is separated by HPLC.

Step 6. Preparation of 4-Bromomethyl-6-chloro-5-propyl-pyrimidine

Br₂ (0.153 ml, 3 mmol) is added dropwise to a solution of4-chloro-6-methyl-5-propyl-pyrimidine (3 mmol) heated at 85° C. in HOAc(10 ml). After the addition, the mixture is stirred at 85° C. for 1hour. The solvent is removed in vacuo and the residue dissolved in EtOAc(15 ml), washed with Na₂S₂O₃ solution (sat. 5 ml), followed by NaHCO₃(10 ml), and brine (10 ml). The organic phase is dried (Na₂SO₄) andevaporated. The resulting yellow oil is purified by flash columnchromatography, which provides the title compound.

Step 7. Preparation of(6-chloro-5-propyl-pyrimidin-4-ylmethyl)-(3-methyl-butyl)-amine

To a stirred solution of 4-bromomethyl-5-propyl-6-chloro-pyrimidine (10mmol) and isoamylamine (4.35 g, 50 mmol) in acetonitrile (30 mL) isadded excess K₂CO₃ (6.9 g). The mixture is stirred at room temperatureovernight. The solvent is removed in vacuo and water (10mL) and DCM (15mL) are added. The layers are separated and the aqueous layer isextracted with DCM (2×15 mL). The combined extracts are washed withbrine (10 ML) and dried (Na₂SO₄) and solvent evaporated to provide thetitle product as an oil.

Step 8. Preparation of 6-Fluoro-pyridine-2-carboxylic Acid(6-chloro-5-propyl-pyrimidin-4-ylmethyl)-(3-methyl-butyl)-amide

To a stirred solution of(6-chloro-5-propyl-pyrimidin-4-ylmethyl)-(3-methyl-butyl)-amine (0.715g, 2.8 mmol) and 6-fluoro-pyridine-2-carboxylic acid (0.47 g, 3.34 mmol)in DCM (10 ml) is added EDCI (0.61 g) and DMAP (0.153 g). The mixture isstirred at room temperature overnight. DCM (10 ml) is added to dilutethe mixture. The mixture is washed with water (5 mL), dried (Na₂SO₄) andsolvent evaporated. Preparative TLC purification of the residue (2:1 ofhexane: ethyl acetate) provides the title product. H¹ NMR δ (CDCl3) 8.69and 8.75 (s, 1H), 7.71-7.97 (m,1H), 7.58-60 (m, 1H), 4.79 and 5.07 (s,2H), 3.50-3.64 (m, 21), 2.59-2.87 (m, 2H), 1.45-1.72 (m,5H), 0.92-1.09(m, 4H), 0.81 (d, 6H).

B.N-(6-Chloro-5-propyl-pyrimidin-4-ylmethyl)-N-ethyl-2,5-difluoro-benzamide

This compound is prepared essentially as described above. LCMS: M+1354.04.C.2,5-Difluoro-N-idobutyl-N-(8-propyl-[1,2,4]triazolo[1,5-c]pyrimidin-7-ylmethyl)-benzamide

Step 1. Preparation of 5-propyl-6-methyl-pyrimidin-4-one

NaOMe (1.30 g, 24 mmol) is added to a stirred solution of formamidine(12 mmol) in MeOH (75 ml) at room temperature. The mixture is stirredfor 15 minutes. 2-Acetyl-pentanoic acid methyl ester (10 mmol) is addedand the mixture is stirred at room temperature overnight. Acetic acid(0.72 g, 12 mmol) is added and the solvent is removed in vacuo. Water(30 ml) is added to the residue and it is extracted with 2-butanone(3×30 ml). The combined extracts are washed with brine (40 ml), dried(Na₂SO₄), and evaporated, to provide a yellow solid, which is used inthe next step without further purification.

Step 2. Preparation of 5-propyl4-chloro-6-methyl-pyrimidine

A mixture of 5-propyl-6-methyl-pyrimidin4-one (10 mmol) and POCl₃ (25ml) is heated at 85° C. for 4 hours. The solvent is removed in vacuo andEtOAc (30 ml) and water (30 ml) are added to the residue. NaHCO₃ iscarefully added until the pH of aqueous layer is greater than 7. Thelayers are separated and the aqueous layer is extracted with EtOAc (2×30ml). The combined extracts are washed with brine (50 ml), dried(Na₂SO₄), and evaporated. Flash column purification of the residue with6:1 EtOAc:hexane provides the title product as a light yellow oil.

Step 3. Preparation of (6-Methyl-5-propyl-pyrimidin-4-yl)-hydrazine

A mixture of 5-propyl-4-chloro-6-methyl-pyrimidine (10 mmol) andhydrazine monohydrate (30 mmol) in ethanol (20 mL) is heated at 80° C.overnight. Solvent is removed in vacuo and the residue solid is in thenext step without further purification.

Step 4. Preparation of7-Methyl-8-propyl-[1,2,4]triazolo[1,5-c]pyrimidine

A solution of (6-methyl-5-propyl-pyrimidinyl)-hydrazine (10 mmol) informic acid (3 mL) is heated at 110° C. overnight. The excess amount offormic acid is removed in vacua. To the residue is added EtOAc (15 mL)and the mixture is washed with saturated sodium bicarbonate solution.The layers are separated and the aqueous layer is extracted with EtOAc(2×15 mL), dried and solvent removed. PTLC separation (5% methanol inmethylene chloride) gives the title compound.

Step 5. Preparation of7-Bromomethyl-8-propyl-[1,2,4]triazolo[1,5-c]pyrimidine

Br₂ (0.153 ml, 3 mmol) is added dropwise to a solution of7-methyl-8-propyl-[1,2,4]triazolo[1,5-c]pyrimidine (3 mmol) heated at100° C. in HOAc (10 ml). After the addition, the mixture is stirred at100° C. overnight. The solvent is removed in vacuo and the residuedissolved in EtOAc (15 ml), washed with Na₂S₂O₃ solution (sat. 5 ml),followed by NaHCO₃ (10 ml), and brine (10 ml). The organic phase isdried (Na₂SO₄) and evaporated. The resulting yellow oil is purified byPTLC, which provides the title compound.

Step 6. Preparation ofIsobutyl-(8-propyl-[1,2,4]triazolo[1,5-c]pyrimidin-7-ylmethyl)-amine

To a solution of 7-bromomethyl-8-propyl-[1,2,4]triazolo[1,5-c]pyrimidine(16.58 mmol) in CH₃CN (30 ml) is added K₂CO₃ (9.15 g, 66.3 mmol),isobutylamine (6.6 ml, 66.3 mmol) and the mixture is stirred at roomtemperature overnight. The solvent is removed in vacuo and to theresidue is added water (60 ml) and EtOAc (60 ml). The layers areseparated and the organic layer is washed with brine (20 ml) and dried(Na₂SO₄). Evaporation of the solvent provides a light yellow oil, whichis used in next step without further purification.

Step 7. Preparation of 6-Fluoro-pyridine-2-carboxylic acidisobutyl-(8-propyl[1,2,4]triazolo-[1,5-c]pyrimidin-7-ylmethyl)-amide

To a stirred solution ofisobutyl-(8-propyl-[1,2,4]triazolo[1,5-c]pyrimidin-7-ylmethyl)-amine(2.8 mmol) and 6-fluoro-pyridine-2-carboxylic acid (0.47g, 3.34 mmol) inDCM (10 ml) is added EDCI (0.61 g) and DMAP (0.153 g). The mixture isstirred at room temperature overnight. DCM (10 ml) is added to dilutethe mixture. The mixture is washed with water (5 mL), dried (Na₂SO₄) andsolvent evaporated. Preparative TLC purification of the residue (2:1 ofhexane: ethyl acetate) provides the title product. H¹ NMR δ (CDCl3)(mixture of rotamers) 9.25 (s, 0.51), 9.18 (s, 0.5H), 8.39 (s, 0.5H),8.36 (s, 0.5H), 7.78-7.93 (m, 1H), 7.68 (d, 0.5H), 7.57 (d, 0.5H), 6.99(d, 0.5H), 6.92 (d, 0.5H), 5.03 (s, 1H), 4.89 (s, 1H), 3.52 (d, 1H),3.35 (d, 1H), 3.12 (t, 1H), 2.83 (t, 1H), 1.95-2.20 (m, 1H), 1.59-1.85(m, 2H), 0.88-1.10 (m, 6H), 0.82 (t, 3H).

D.2,5-Difluoro-N-isobutyl-N-(8-propyl-[1,2,4]triazolo[1,5-c]pyrimidin-7-ylmethyl)-benzamide

This compound is prepared essentially as described above. H¹ NMR δ(CDCl3) (mixture of rotamers) 9.24 (s, 1H), 8.39 (s, 1H), 7.00-7.25 (m,3H), 4.88 (s, 1H), 4.59 (s, 1H), 3.22 (d, 2H), 3.15 (t, 1H), 2.81 (t,1H), 1.98-2.18 (m, 1H), 1.73-2.18 (m, 1H), 1.53-1.66 (m, 1H), 0.80-1.12(m, 9H).

E. Additional Representative Aryl Acid Pyrimidinyl Methyl Amides,Pyridazinyl Methyl Amides and Related Compounds

The following compounds are prepared via the methods set forth above.

Example 3 Ligand Binding Assay

The high affinity of compounds of this invention for the benzodiazepinesite of the GABA_(A) receptor is confirmed using a binding assayessentially described by Thomas and Tallman (1981) J. Bio. Chem.156:9838-9842, and (1983) J. Neurosci. 3:433-440).

Rat cortical tissue is dissected and homogenized in 25 volumes (w/v) ofBuffer A (0.05 M Tris HCl buffer, pH 7.4 at 4° C.). The tissuehomogenate is centrifuged in the cold (4° C.) at 20,000×g for 20minutes. The supernatant is decanted, the pellet rehomogenized in thesame volume of buffer, and centrifuged again at 20,000×g. Thesupernatant of this centrifugation step is decanted and the pelletstored at −20° C. overnight. The pellet is then thawed and resuspendedin 25 volumes of Buffer A (original wt/vol), centrifuged at 20,000×g andthe supernatant decanted. This wash step is repeated once. The pellet isfinally resuspended in 50 volumes of Buffer A.

Incubations contain 100 μl of tissue homogenate, 100 μl of radioligand,(0.5 nM ³H-RO15-1788 [³H-Flumazenil], specific activity 80 Ci/mmol), andtest compound or control (see below), and are brought to a total volumeof 500 μl with Buffer A. Incubations are carried out for 30 minutes at4° C. and then rapidly filtered through Whatman GFB filters to separatefree and bound ligand. Filters are washed twice with fresh Buffer A andcounted in a liquid scintillation counter. Nonspecific binding (control)is determined by displacement of ³H RO15-1788 with 10 μM Diazepam(Research Biochemicals International, Natick, Mass.). Data are collectedin triplicate, averaged, and percent inhibition of total specificbinding (Total Specific Binding=Total−Nonspecific) is calculated foreach compound.

A competition binding curve is obtained with up to 11 points spanningthe compound concentration range from 10⁻¹²M to 10⁻⁵M obtained per curveby the method described above for determining percent inhibition. K_(i)values are calculated according the Cheng-Prussof equation. Each of thecompounds set forth above was tested in this fashion and each was foundto have a K_(i) of <1 μM. Preferred compounds of the invention exhibitK_(i) values of less than 100 nM and more preferred compounds of theinvention exhibit K_(i) values of less than 10 nM.

Example 4 Electrophysiology

The following assay is used to determine if a compound of the inventionacts as an agonist, an antagonist, or an inverse agonist at thebenzodiazepine site of the GABA_(A) receptor.

Assays are carried out essentially as described in White and Gurley(NeuroReport 6:1313-1316, 1995) and White, Gurley, Hartnett, Stirling,and Gregory (Receptors and Channels 3:1-5, 1995) with modifications.Electrophysiological recordings are carried out using the two electrodevoltage-clamp technique at a membrane holding potential of −70 mV.Xenopus laevisoocytes are enzymatically isolated and injected withnon-polyadenylated cRNA mixed in a ratio of 4:1:4 for α, β and γsubunits, respectively. Of the nine combinations of α, β and γ subunitsdescribed in the White et al. publications, preferred combinations areα₁β₂γ₂, α₂β₃γ₂, α₃β₃γ₂, and α₅β₃γ₂. Preferably all of the subunit cRNAsin each combination are human clones or all are rat clones. The sequenceof each of these cloned subunits is available from GENBANK, e.g., humanα₁, GENBANK accession No. X14766, human α₂, GENBANK accession No.A28100; human α₃, GENBANK accession No. A28102; human α₅, GENBANKaccession No. A28104; human β₂, GENBANK accession No. M82919; human β₃,GENBANK accession No. Z20136; human γ₂, GENBANK accession No. X15376;rat α₁, GENBANK accession No. L08490, rat α₂, GENBANK accession No.L08491; rat α₃, GENBANK accession No. L08492; rat α₅, GENBANK accessionNo. L08494; rat β₂, GENBANK accession No. X15467; rat β₃, GENBANKaccession No. X15468; and rat γ₂, GENBANK accession No. L08497. For eachsubunit combination, sufficient message for each constituent subunit isinjected to provide current amplitudes of >10 nA when 1 μM GABA isapplied.

Compounds are evaluated against a GABA concentration that evokes <10% ofthe maximal evocable GABA current (e.g., 1 μM-9 μM). Each oocyte isexposed to increasing concentrations of a compound being evaluated (testcompound) in order to evaluate a concentration/effect relationship. Testcompound efficacy is calculated as a percent-change in currentamplitude: 100*((Ic/I)-1), where Ic is the GABA evoked current amplitudeobserved in the presence of test compound and I is the GABA evokedcurrent amplitude observed in the absence of the test compound.

Specificity of a test compound for the benzodiazepine site is determinedfollowing completion of a concentration/effect curve. After washing theoocyte sufficiently to remove previously applied test compound, theoocyte is exposed to GABA+1 μM RO15-1788, followed by exposure to GABA+1μM RO15-1788+test compound. Percent change due to addition of compoundis calculated as described above. Any percent change observed in thepresence of RO15-1788 is subtracted from the percent changes in currentamplitude observed in the absence of 1 μM RO15-1788. These net valuesare used for the calculation of average efficacy and EC₅₀ values bystandard methods. To evaluate average efficacy and EC₅₀ values, theconcentration/effect data are averaged across cells and fit to thelogistic equation.

Example 5 MDCK Toxicity Assay

This Example illustrates the evaluation of compound toxicity using aMadin Darby canine kidney (MDCK) cell cytotoxicity assay.

1 μL of test compound is added to each well of a clear bottom 96-wellplate (PACKARD, Meriden, Conn.) to give final concentration of compoundin the assay of 10 micromolar, 100 micromolar or 200 micromolar. Solventwithout test compound is added to control wells.

MDCK cells, ATCC No. CCL-34 (American Type Culture Collection, Manassas,Va.), are maintained in sterile conditions following the instructions inthe ATCC production information sheet. Confluent MDCK cells aretrypsinized, harvested, and diluted to a concentration of 0.1×10⁶cells/mL with warm (37° C.) medium (VITACELL Minimum Essential MediumEagle, ATCC catalog # 30-2003). 100 μL of diluted cells is added to eachwell, except for five standard curve control wells that contain 100 μLof warm medium without cells. The plate is then incubated at 37° C.under 95% O₂, 5% CO₂ for 2 hours with constant shaking. Afterincubation, 50 μL of mammalian cell lysis solution (from the PACKARD(Meriden, CT) ATP-LITE-M Luminescent ATP detection kit) is added perwell, the wells are covered with PACKARD TOPSEAL stickers, and platesare shaken at approximately 700 rpm on a suitable shaker for 2 minutes.

Compounds causing toxicity will decrease ATP production, relative tountreated cells. The ATP-LITE-M Luminescent ATP detection kit isgenerally used according to the manufacturer's instructions to measureATP production in treated and untreated MDCK cells. PACKARD ATP LITE-Mreagents are allowed to equilibrate to room temperature. Onceequilibrated, the lyophilized substrate solution is reconstituted in 5.5mL of substrate buffer solution (from kit). Lyophilized ATP standardsolution is reconstituted in deionized water to give a 10 mM stock. Forthe five control wells, 10 μL of serially diluted PACKARD standard isadded to each of the standard curve control wells to yield a finalconcentration in each subsequent well of 200 nM, 100 nM, 50 nM, 25 nMand 12.5 nM. PACKARD substrate solution (50 μL) is added to all wells,which are then covered, and the plates are shaken at approximately 700rpm on a suitable shaker for 2 minutes. A white PACKARD sticker isattached to the bottom of each plate and samples are dark adapted bywrapping plates in foil and placing in the dark for 10 minutes.Luminescence is then measured at 22° C. using a luminescence counter(e.g., PACKARD TOPCOUNT Microplate Scintillation and LuminescenceCounter or TECAN SPECTRAFLUOR PLUS), and ATP levels calculated from thestandard curve. ATP levels in cells treated with test compound(s) arecompared to the levels determined for untreated cells. Cells treatedwith 10 μM of a preferred test compound exhibit ATP levels that are atleast 80%, preferably at least 90%, of the untreated cells. When a 100μM concentration of the test compound is used, cells treated withpreferred test compounds exhibit ATP levels that are at least 50%,preferably at least 80%, of the ATP levels detected in untreated cells.

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein: Ar representsphenyl, naphthyl or a 5- to 10-membered heteroaryl group, each of whichis substituted with from 0 to 4 groups independently selected from R₈;R₁ is independently chosen at each occurrence from: (a) hydrogen,halogen, nitro and cyano; and (b) groups of the formula:

 wherein: G is a bond, C₁—C₄alkyl, —N(R_(B))—, —O—, —C(═O)—,—C(═O)N(R_(B))—, —N(R_(B))C(═O)—, —S(O)_(m)—, —CH₂C(═O)—,—S(O)_(m)N(R_(B))— or —N(R_(B))S(O)_(m)—; wherein m is 0, 1 or 2; andR_(A) and each R_(B) are independently selected from: (i) hydrogen; and(ii) C₁-C₈alkyl, C₂-C₈alkenyl, (C₃-C₈carbocycle)C₀-C₄alkyl and (3- to8-membered heterocycle)C₀-C₄alkyl, each of which is substituted withfrom 0 to 4 substituents independently selected from halogen, hydroxy,nitro, cyano, amino, C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₄alkanoyl, mono- anddi(C₁-C₄alkyl)amino, C₁-C₄haloalkyl and C₁-C₄haloalkoxy; R₄ is hydroxy,nitro, cyano, amino, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,C₃-C₇cycloalkyl, C₁-C₈haloalkyl, C₁-C₈alkoxy, C₁-C₈haloalkoxy,C₂-C₈alkyl ether, C₂-C₈haloalalkyl ether, or mono- ordi-(C₁-C₈alkyl)amino(C₀-C₄alkyl); R₅ and R₆ are independently hydrogen,methyl or ethyl; R₇ represents C₁-C₈alkyl, C₂-C₈alkenyl,(C₃-C₇cycloalkyl)C₀-C₄alkyl, or benzyl that is substituted with from 0to 3 substituents independently chosen from halogen, nitro,trifluoromethyl, trifluoromethoxy, cyano and hydroxy; and R₈ isindependently selected at each occurrence from halogen, hydroxy, nitro,cyano, amino, C₁-C₈alkyl, C₂-C₈alkenyl, C₁-C₈alkynyl,(C₃-C₇cycloalkyl)C₀-C₈alkyl, C₁-C₈haloalkyl, C₁-C₈alkoxy,(C₃-C₇cycloalkyl)C₁-C₈alkoxy, C₁-C₈haloalkoxy, C₂-C₈alkyl ether,C₃-C₈alkanone, C₁-C₈alkanoyl, (3- to 7-memberedheterocycloalkyl)C₀-C₈alkyl, C₁-C₈hydroxyalkyl, C₁-C₈aminoalkyl, andmono- and di-(C₁-C₈alkyl)aminoC₀-C₈alkyl.
 2. A compound or saltaccording to claim 1, wherein Ar is phenyl or pyridyl, each of which issubstituted with from 0 to 4 substituents independently selected fromR₈.
 3. A compound or salt according to claim 1, wherein each R₈ isindependently chosen from halogen, hydroxy, amino, cyano, C₁-C₄alkyl,C₁-C₄alkoxy, mono- and di-C₁-C₄alkylamino, C₂-C₄alkanoyl,(C₃-C₇cycloalkyl)C₀-C₂alkyl, C₁-C₂haloalkyl and C₁-C₂haloalkoxy.
 4. Acompound or salt according to claim 2, wherein Ar is phenyl or2-pyridyl, each of which is substituted with from 0 to 3 substituentsindependently chosen from chloro, fluoro, hydroxy, cyano, amino,C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₂alkylamino, C₁-C₂haloalkyl andC₁-C₂haloalkoxy.
 5. A compound or salt according to claim 4, wherein Aris phenyl or 2-pyridyl, each of which is substituted with 1, 2 or 3substituents independently chosen from fluoro and chloro.
 6. A compoundor salt to claim 4, wherein Ar is 2,6-difluorophenyl or6-fluoro-pyridin-2-yl.
 7. A compound or salt according to claim 1,wherein R₄ is hydroxy, cyano, amino, C₁-C₈alkyl, C₂-C₈alkenyl,C₂-C₈alkynyl, C₁-C₈alkoxy, C₂-C₈alkyl ether, or mono- ordi-(C₁-C₈alkyl)amino(C₀-C₄alkyl).
 8. A compound or salt according toclaim 7, wherein R₄ is C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl orC₁-C₆alkoxy.
 9. A compound or salt according to claim 1, wherein each R₁is independently chosen from: (a) hydrogen and halogen; and (b) groupsof the formula:

wherein: G is a bond, —NH—, —N(R_(B))—, —O— or —C(═O)—; and R_(A) andR_(B) are independently selected from: (i) hydrogen; and (ii)C₁-C₆alkyl, C₂-C₆alkenyl and (C₃-C₇cycloalkyl)C₀-C₂alkyl, each of whichsubstituted with from 0 to 4 substituents independently selected fromhydroxy, halogen, cyano, amino, C₁-C₂alkyl and C₁-C₂alkoxy.
 10. Acompound or salt according to claim 9 wherein each R₁ is independentlyselected from hydrogen, hydroxy, halogen, C₁-C₆alkyl, C₁-C₆alkoxy,C₂-C₄alkanoyl, C₁-C₂haloalkyl, C₁-C₂haloalkoxy, and mono- anddi-(C₁-C₄alkyl) amino.
 11. A compound or salt according to claim 10,wherein: each R₁ is independently chosen from hydrogen, halogen,C₁-C₆alkyl and C₁-C₆alkoxy; and R₄ is C₁-C₆alkyl or C₁-C₆alkoxy.
 12. Acompound or salt according to claim 11, wherein Ar is phenyl or pyridyl,each of which is substituted with from 0 to 4 substituents independentlyselected from halogen, hydroxy, amino, cyano, C₁-C₄alkyl, C₁-C₄alkoxy,mono- and di-C₁-C₄alkylamino, C₂-C₄alkanoyl,(C₃-C₇cycloalkyl)C₀-C₂alkyl, C₁-C₂haloalkyl and C₁-C₂haloalkoxy.
 13. Acompound or salt according to claim 12, wherein Ar is phenyl or2-pyridyl, each of which is substituted with 1, 2 or 3 substituentsindependently chosen from fluoro and chloro.
 14. A compound or saltaccording to claim 13, wherein Ar is 2,6-difluorophenyl or6-fluoro-pyridin-2-yl.
 15. A compound or salt according to claim 1,wherein R₅ and R₆ are both hydrogen.
 16. A compound or salt according toclaim 1 wherein R₇ is C₃-C₆alkyl.
 17. A compound or salt according toclaim 16 wherein R₇ is 3methyl-butyl, isobutyl or n-butyl.
 18. Acompound or salt according to claim 1, wherein the compound is 2,5-Difluoro-N-isobutyl-N-(8-propyl-[1,2,4]triazolo,[1,5-c]pyrimidin-7-ylmethyl)-benzamide.19. A compound or salt according to claim 1, wherein the compoundexhibits a K_(i) of 1 micromolar or less in an assay of GABA_(A)receptor binding.
 20. A pharmaceutical composition comprising a compoundor salt according to claim 1 in combination with a physiologicallyacceptable carrier or excipient.
 21. A pharmaceutical compositionaccording to claim 20, wherein the pharmaceutical composition isformulated as an injectible fluid, an aerosol, a cream, a gel, a pill, acapsule, a syrup or a transdermal patch.
 22. A packaged pharmaceuticalpreparation comprising a pharmaceutical composition according to claim20 in a container and instructions for using the composition to treat apatient suffering from a sleep disorder.
 23. A method for the treatmentof a sleep disorder, comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of a compound or saltaccording to claim 1.